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Hydroshear Plus User Guide

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1. 40 40 INUMBER S o 41 SPEED E 41 EDITING BASIC SHEARING PARAMETERG cccscsccssscccssscccscccccscccccsccccccccceccccccccccscccccscsccsecees 41 FINDING THE MAXIMUM SPEED CODE cccccocssssccssscccccccccscccccscccccsccccsccccccscccscccccscccccscscesecees 41 STEP DOWN SHEARING METHOD 42 iii DIGILAB 4 3 7 SETTING UP A WASH SCHEME 43 4 3 8 SAVING SHEARING PARAMETERS VALUES TO A FILE eessesssessesosesssossosssossesoscssssosesssossosssosse 45 4 3 9 LOADING SAVED SHEARING PARAMETERS FROM A FILE cccccssccccscccsscccccccccccccccccccccscsccees 46 4 3 10 SAVING NEW DEFAULT SHEARING PARAMETER VALUES ssssssessscssesssossecosessssosesssossesssosse 47 4 3 11 RESTORING SHEARING PARAMETER DEFAULT VALUEG cccccccscccssccccsccccccccccccccccccccccccceees 48 4 4 OPT RATION e HU EC ETE 48 4 5 VIE VE TTE 49 49 452 RUNSIATUS 49 4 5 3 CURRENT PUMP VOLUME uese thid bt genae o 50 4 5 4 CURRENT VALVE POSITION e
2. 73 1V DIGILAB 8 1 2 CLOGGED SHEARING ASSEMBLY essssesssecsssosssesssessssosssesosessssessessssessssessseosseosssesseessssesssessssoss 73 8 1 3 DECREASING SAMPLE VOLUME 74 8 1 4 INCREASING SAMPLE VOLUME essesssessecseesssessesssesseossesosossesseoseessessseossesseesseossesssossesssessessseso 74 8 2 SAMPLE AND FRAGMENT PROBLEMS eesssessssesssessscosssesseessseosssesssesosesosesoseessseosssesssessssosssessse 74 SA 74 8 2 2 SIZE OF SHEARED DNA FRAGMENTS IS INCORRECT seessseosssesssessseosssesssessscosssesssessssesseessseoso 74 8 2 3 THE FRAGMENTS PRODUCED DO NOT CLONE WELL ssesssessssesssesssessssesssecossosssesssesssssossessseoso 75 8 3 SOFTWARE ERROR 76 9 5 1 ERROR 37 AT SERIAL PORT aa oa aaa 76 8 3 2 MACHINE PARAMETERS 76 93 3 SYRINGE VOLUME 77 1 77 539 REIRACTION SPEED CONSE Te V VEU 77 5 50 78 78 8 3 8 SETTING NEW DEFAULT MACHINE PARAMETER VALUE ccccosscccssccccscccccccccccccsccccccccccccees 79 8 3 9 RLAD TIMEOUT ERROR 80 8 3 10 PUMP OVERLOAD OR PLUNGER OVERLOAD ERROR sesssessesssessssssosssossesssossscssesssossessesssesssoso 80 8 3 11 OTHER ERROR MESSAGES eeosesesssessscosssessseosseosssesosessssosssossesssseosssesssessssesssesssesossesssesss
3. MyIDM E 73 Program Files Qoobox C3 SwDist 8 temp WINDOWS 2 Driver D UISB2 0 EASY TRANSFER LINK CABLE FOF USB 2 0 Easy Link Met Cable 8 O USB 2 0 Network Cable USB 2 0 TO R5232 Cable B USB 2 0 TO R5232 Converter O USB Link Cable USB Network Cable USB TO 2 x 5232 Cable Linux mac manual E windows USB TO 2 x R5422 Cable 3 USB TO Parallel Printer Cable x Files Currently on the CD manual lt MEK 4 5 windows After double clicking on windows another screen will appear as shown below To start to load the cable drivers double click on Setup Hydroshear Plus User Manual Page 17 DIG L B Chapter 3 Installing the Hydroshear Plus File Edit View Favorites Tools Help lt Back gt 27 Search D Folders Address D USB TO 2 x R5232 Cable windows Folders x Files Currently on the CD O drivers E EXACT 1 cJ cJ E F48ad320f524827469bd79b72F i WINZK amp XP WINSS8ME Customize Customize MaosUnst Setup i386 zx a a 3 MyIDM Program Files Qoobox SWDist temp WINDOWS 1 Driver D LISB2 0 EASY TRANSFER CABLE FOF USB 2 0 Easy Link Net Cable USB 2 0 Network Cable USB 2 0 TO R5232 Cable 5 USB 2 0 TO R5232 Converter USB Link Cable USB Network Cable USB TO 2 x R5232
4. 1 VS 12 CHAPTER3 INSTALLING THE HYDROSHEAR PLUS FINDING A LOCATION FOR THE HYDROSHEAR PLUG cccccssccsssccsscccsscccsccccccccsccccscccceccceescees 13 SOFTWARE INSTALLATION reU 13 HXDROSIEAR IS eoe ei a E 13 CABLE DRIVER SOFTWARE PEE OE a 16 HARDWARE TEE 26 EID B 27 SYRINGE INSTALLATION EERO 28 SHEARING ASSEMBLY INSTALLATION eoe es ee asks eun as 3l DESCRIPTION OF SHEARING ASSEMBLY COMPONENTS cccccccsscccsccccccccccccccsccccsccccsccscccccscees 31 DETERMINING THE ORIENTATION OF AN UNLABELED ORIFICE BOX eee ee eee ee eeu 33 ATTACHING THE SHEARING ASSEMBLY TO THE HYDROSHEAR PLUS 34 CALIBRATING THE SHEARING 35 4 USING THE HYDROSHEAR PLUS SOFTWARE 4 1 4 2 4 3 4 3 1 4 3 2 4 3 3 4 3 4 4 3 5 4 3 6 STARTING THE HYDROSHEAR PLUS SOFTWARE cccccccssscccssscccscsccccscccscccccsccccsccccscccccccccesees 37 OVERVIEW OF THE MAIN CONTROL PANEL cccccccssssccccscccccccccccccscccccccsccccccccccccccccccccscccsceees 39 SHEARING PARAMETERS
5. Ej machineDeFaulEs File name My Shearing Run Save as Jal Files 3 Navigate to the location where you would like to save the file 4 a name for the file in the text box 5 Click the SAVE button 6 The new shearing parameters values are saved with the specified name in the specified location Hydroshear Plus User Manual Page 45 DIGIL B 4 3 9 LOADING SAVED SHEARING PARAMETERS FROM A FILE Chapter 4 Using the Hydroshear Plus Software To load custom shearing parameters that had been previously saved to a file 1 Click on the Read from File button in the Shearing Parameters box of the Hydroshear Plus Main Panel 2 the dialog box that opens select the desired settings file and click on Open Choose file to read 2 x Look in Hydroshearzt3 ES m 09 exe userDefaults iwen lem machineDefaults My Shearing Run Settings File name My Shearing Aun Settings Files of type A Files Cancel 3 Another window appears allowing you to verify that settings are correct Parameters In File ash Solution I ash Solution II ash Solution isie laie iti Laie e 5 5 Use These Values Cancel 4 these settings are correct click on the Use These Values button If not click on Cancel Note that even though the settings in this dialog box can be ch
6. Reinitializing the syringe pump To reach the Manual Operation window click the MANUAL OPERATION button on the Hydroshear Plus software main panel This will open the Manual Operation control window Manual Operation Reinitialize Pump 6 1 EXPLANATION OF MANUAL OPERATION CONTROLS Reinitialize Pump This button causes the Hydroshear Plus syringe pump to reset to the zero position at the top of its range of movement This is essential for the syringe to be able to accurately aspirate and dispense the correct amount of fluid Note that the syringe pump should always be reinitialized after any manual operations have been performed Hydroshear Plus User Manual Page 60 DIGIL B Chapter 6 Manual Operation Volume This box allows users to enter a volume that will be either aspirated into or expelled from the Hydroshear Plus syringe A positive value will cause fluid or air to be drawn into the syringe To expel a certain volume from the syringe precede the value with a negative sign Speed Code The speed code can range from 0 to 40 with an increasing speed code value corresponding with a decreased plunger speed A speed code of 0 not recommended for actual shearing runs results in the fastest speed 1 2 seconds per stroke while a speed code of 40 results in the slowest speed 600 seconds per stroke Valve Position The valve position choices are Input Output Wash 1 Wash 2 and Wash 3 amp Closed
7. TO THE PLACE YOU OBTAINED THEM ALL RETURNS SHALL BE SUBJECT TO NI S THEN CURRENT RETURN POLICY Definitions As used in this Agreement the following terms have the following meanings wv do not accept the License Agreement s Page 14 DIG L B Chapter 3 Installing the Hydroshear Plus 5 Click Next to start the installation HydroShear Start Installation Review the following summary before continuing Adding or Changing HydroShear Files NIMIS 4 1 Run Time Support Click the Next button to begin installation Click the Back button to change the installation settings Sese es 6 Click Finish to close this dialog box and restart the computer to finalize the installation procedure HydroShear Installation Complete The installer has finished updating your system lt lt Back Finish Hydroshear Plus User Manual Page 15 DIG L B Chapter 3 Installing the Hydroshear Plus 3 2 2 CABLE DRIVER SOFTWARE The Hydroshear Plus is shipped with a USB to 2X Serial Connector cable Unless the Hydroshear Plus 1 purchased with a laptop PC from Digilab the proper drivers need to be installed prior to connecting cable to the Hydroshear Plus Load the CD into the CD Drive of the PC being used for the Hydroshear Plus Right click on the Start button at the bottom left hand corner of the toolbar and click on Explore Scroll to the CD drive as listed in the Explor
8. 17 19 500bp ladder 5000 bp 3500 bp 2500 bp 2000 bp 1500 bp CO ON OQ A 1000 bp m wo om 1 agarose gel run at 145V for 2 hours All samples are 2 ug 100 ul lambda Figure 9 1 Correlation of speed code with DNA fragment size Hydroshear Plus User Manual Page 82 DIG L B Chapter 9 Performance Data 9 2 SMALLEST STARTING FRAGMENT SIZE THAT CAN BE SHEARED The Hydroshear Plus shears nearly all DNA in a sample of 4kb linear fragments The Hydroshear Plus can shear a sample of 3kb linear fragments but some DNA 1s left unsheared Method Samples of varying fragment sizes ranging from 3 kb to 10 kb were sheared at speed code 3 using a standard shearing assembly The sheared samples were then run on an agarose gel alongside unsheared samples of the same size as the starting material Result With starting material of 4kb or greater nearly the entire DNA sample is sheared With a 3kb starting fragment sample shearing does occur but some of the DNA in the sample remains unsheared 10 11 12 13 14 S ample Sheared 3 kb sample Unsheared 3 kb sample Sheared 4 kb sample Unsheared 4 kb sample Sheared 5 kb sample Unsheared 5 kb sample Sheared 6 kb sample 2 3 4 5 6 7 amp 9 Unsheared 6 kb sample Sheared 8 kb sample Unsheared 8 kb sample Sheared 10 kb sample 1 agarose gel run at 105V for 1 hour Unsheared 10 kb sample All samples gel extracted from a 1
9. 7 Click the Start button 8 One pump pass is conducted at the specified speed code and the specified volume is drawn into the syringe Hydroshear Plus User Manual Page 62 DIG L B Chapter 6 Manual Operation 6 3 MANUAL EJECTION FROM THE HYDROSHEAR PLUS SYRINGE The following procedure should not be used when the syringe 1 empty To eject a volume of fluid or air from the syringe follow these steps 1 Locate the Status box on right hand side of the Hydroshear Plus software main window 2 Examine the Current pump volume text box to determine how much fluid or air is in the Hydroshear Plus syringe 3 Click on the Manual Operation button to open the Manual Operation window 4 Select the valve position to either the INPUT or OUTPUT position depending upon whether you want to eject the sample into the input or output tubing Place an appropriate tube or collection vial if required at the end of the input or output tubing to collect any sample that will be ejected 5 In the Volume box enter a negative value for the volume you wish to eject from the syringe For example if you wish to eject 35041 enter 350 in the Volume box 6 Inthe Speed Code box specify the speed code at which you wish the syringe pump to move Remember the higher the speed code the slower the syringe pump will move For manual operation 10 1s a reasonable speed code Choose 20 if the shearing assembly is clogged or if you
10. 7 2 1 CLEANING WITH WEAK DETERGENT 64 7 2 2 CLEANING WITH WEAK BASE AND ACID IN SEQUENCE ssesssessesosessscssososossesosossssosesssossesssosse 65 7 2 3 CLEANING WITH 10 BLEACH ssescssecsseccseccossosoecsseccoseossecosecossosoecoosecosseossecosseosssossesosseosseossoo 66 7 3 PERIODIC MANTENAN E E E E E EE 67 7 4 UNCLOGGING SHEARING ORIFICE BOXES USING SONICATION sssesssessssesssessssosssesssessseossseo 67 7 5 REPLACING HYDROSHEAR PLUS TUBING eeesssesssessssesssesssessssosssesosessssessesssssosssesssessssosssessse 70 7 5 1 REPLACING THE HYDROSHEAR PLUS OUTPUT TUBING sesssessesssessscssosssossesosessesosesssossesseosse 70 7 5 2 REPLACING THE HYDROSHEAR PLUS INPUT AND WASH TUBING cccccccssccccsccccscccccccees 71 7 5 3 REMOVING THE HYDROSHEAR PLUS SYRINGE e seeosssesssesssessssesssesssessssesssesossossessosesossosssessse 71 7 6 DRIVE SCREW 68608 72 CHAPTERS8 TROUBLESHOOTING 8 1 PROBLEMS OBSERVED DURING SHEARING RUNS eee 73 8 1 1 AIR BUBBLES IN THE SYRINGE eeeeeeceecsessesesse sse
11. Cable Linux mac O manual O windows USB TO 2 x R5422 Cable USB TO Parallel Printer Cable 2 000000 0600 Click Install USB Compound Device Setup Press Install Button to Install the drives For USB Serial Parallel compound device For cancelling the installation press Est button Click Continue Anyway Software Installation AN The software are installing has not passed Windows Logo testing to verify its compatibility with Windows F me why this testing iz important Continuing your installation of this software may impair or destabilize the correct operation of your system either immediately or in the future Microsoft strongly recommends that vou stop this installation now and contact the software vendor for software that has passed Windows Logo testing STOP Installation Hydroshear Plus User Manual Page 18 DIGILAB This window will appear twice so it will be necessary to click Continue Anyway a second time Chapter 3 Installing the Hydroshear Plus The USB cable driver installation is completed Click Exit to complete the process USB Compound Device Setup cg Installation completed Press Exit to terminate the application need to restart the machine Unplug and Replug the device if it is currently connected Plug the USB to RS232 adapter into an available USB port on computer The Welcome to the Found
12. DNA ladder 1 kb DNA ladder cheared about 2ug 100ul Loaded 0 02548 on gel samples sheared at speed 3 Figure 9 2 Determining the lowest starting fragment size Hydroshear Plus User Manual Page 83 DIG L B Chapter 9 Performance Data 9 3 SMALLEST STARTING SAMPLE VOLUME THAT CAN BE SHEARED The Hydroshear Plus can shear a sample volume as small as 40 ul However when such a small sample size 15 used a significant percentage of the sample can be lost due to the dead volume in the system Digilab recommends a sample size of at least 100 Method Samples of varied volumes ranging from 10ul to 100ul were sheared at speed code 10 The sheared samples were then run on an agarose gel Result Sample volumes of 401 or greater resulted in a consistent fragment length range Sample Volume 10 ul 20 ul 30 ul 40 ul 50 ul 60 ul 70 ul 80 ul 90 ul 6 7 8 F l0 11 12 13 1 agarose gel Run at 105 V for 1 hour 100 ul All samples O 0 lug ul lambda sheared at speed 10 F 500 bp ladder Figure 9 3 Determining the lowest possible volume of starting material Hydroshear Plus User Manual Page 84 DIG L B Chapter 9 Performance Data 9 4 SMALLEST FRAGMENTS THE HYDROSHEAR PLUS CAN CONSISTENTLY PRODUCE The Hydroshear Plus can generate fragments in the range of 750 bp to 1 5kb with the majority of fragments centered around 1 kb Method Three samples with a 50 kb initial fragment len
13. allows samples to be sheared see Figure 2 1 How the Hydroshear Plus Works and should be handled with care to minimize the risk of dislodging or damaging the orifice The arrow on the orifice box label should always point towards the clear plastic output tubing If the arrow 1 missing you can examine the inside of the orifice to determine the correct orientation as described 1n Section 3 6 2 Determining the Orientation of an Unlabeled Orifice Box The identification number is useful for keeping calibration records Hydroshear Plus User Manual Page 31 DIG L B Chapter 3 Installing the Hydroshear Plus 5 tubing pressure fitting The pressure fitting consists of a plastic ferrule and a metal ring The input tubing 7 1s inserted through the pressure fitting and attached to the input end of the shearing assembly box by screwing the shearing assembly input tubing nut 6 into the orifice box 6 Input tubing nut This nut screws into the orifice box at the tail of the orifice box s arrow label It attaches the orifice box to the input tubing 7 leading to the base screw 7 Input tubing This inflexible orange PEEK tubing 15 attached to the orifice box via the input tubing nut 6 and is attached to the Hydroshear Plus base unit via the base screw 8 8 screw This 1 16 hex screw attaches the shearing assembly to the Hydroshear Plus base unit The threaded side should be closest to the metal ferrule 7 and sh
14. installation CD floppy disk insert it now What vau want the wizard do C Install from a list ar specific location Advanced Click Next to continue Select the driver in the location C windows inf oem22 inf and click Next Found Mew Hardware Wizard Please select the best match for your hardware from the list below 7 LIS B En Serial Port Driver Location ip Semiconductor Technology windowsin oeme inf Semiconductor Technology d usb to 2 s rs232 cableswindewswinSBsme masus This driver is not digitally signed Tell me why driver signing is important Hydroshear Plus User Manual Page 22 DIGILAB Chapter 3 Installing the Hydroshear Plus Click Continue Anyway Hardware Installation A The software you are installing For this hardware LISB ta Serial Port Driver has not passed Windows Logo testing to verify its compatibility with Windows P Tell me why this testing is important Continuing your installation of this software may impair or destabilize the correct operation of your system either immediately or in the future Microsoft strongly recommends that you stop this installation now and contact the hardware vendor for software that has passed Windows Logo testing Continue Anyway i Click Finish to complete the installation Found New Hardware Wizard Completing the Found New Hardware Wizard The wand hes installing the s
15. into place Syringe Mount Screw 9 Pull the plunger down until it is lined up with the pump bracket 10 Replace the syringe mount screw through the syringe plunger and tighten into the pump bracket 11 The syringe should now be aligned correctly and is ready for shearing Hydroshear Plus User Manual Page 30 DIG L B Chapter 3 Installing the Hydroshear Plus 3 6 SHEARING ASSEMBLY INSTALLATION The shearing assembly must be oriented correctly both to prevent irreversible damage to the orifice and to function properly Refer to Figure 3 2 and the instructions below to ensure that you install the shearing assembly correctly 3 6 1 DESCRIPTION OF SHEARING ASSEMBLY COMPONENTS Figure 3 2 Shearing assembly components 1 Clear plastic output tubing The sheared samples and used wash solution will be dispensed from this tubing 2 Output tubing nut This nut screws into the orifice box at the head of the arrow label and attaches the output tubing 1 to the shearing orifice box 4 3 Output tubing plastic ferrule The output tubing 1 is inserted through the ferrule 3 with the tapered end of the ferrule pointing toward the output tubing nut 2 When putting together a shearing assembly the tubing should be flush to the flat end of the ferrule to ensure a good seal when the tubing nut is screwed into the orifice box 4 Orifice box The orifice box contains the high precision ruby and narrow gap that
16. new Hardware Wizard will appear Select No Not this time and click Next Found Mew Hardware Wizard Welcome the Found New Hardware Wizard Windows will search Far current and updated software by looking en your computer the hardware installation or on the windows Update Web site with pour permission Head our privacy policy Can Windows connect to Windows Update to search For software CO this time only CO Yes now and every time connect device Click Hest to continue Hydroshear Plus User Manual Page 19 DIG L B Chapter 3 Installing the Hydroshear Plus Select the radio button Install the software automatically and click Next to continue Found New Hardware Wizard This wizard helps you install software for USB Compound Device If vour hardware came with installation CD floppy disk insert it now What vau want the wizard do C Install from a list ar specific location Advanced Click Next to continue Click Continue Anyway to continue installation Hardware Installation The software you are installing for this hardware USB Compound Device has not passed Windows Logo testing to verify its compatibility with Windows me why this testing is important Continuing your installation of this software may impair or destabilize the correct operation of your system either immediately or in the future
17. screw connection with the 1 4 hex wrench Do not over tighten 4 Ifthe shearing assembly is new it will need to be calibrated as described in following section Each new shearing assembly must be individually calibrated even if the shearing assembly 1 the same type as had been installed previously Hydroshear Plus User Manual Page 34 DIGIL B Chapter 3 Installing the Hydroshear Plus 3 6 4 CALIBRATING THE SHEARING ASSEMBLY Each new shearing assembly needs to be individually calibrated Since the size of each shearing assembly orifice 1s slightly different the same speed code when used with different shearing assemblies may not result in the same fragment size This calibration will determine the speed code to fragment size correlation for a specific shearing assembly 1 Note the identification number on the shearing assembly orifice box This will be used to match the calibration data with the assembly 2 Shear several DNA samples at various speed code values keeping the volume the number of cycles and the concentration constant 3 Shearing at four different speed codes should be sufficient for accurate calibration but we recommend 6 or more for increased precision 4 samples on an agarose gel and make a note of the average fragment size produced at each speed code NOTE Figure 3 4 on the following page shows an example of a gel electrophoretic separation of samples of DNA sheared wi
18. syringe with Wash Solution 3 for at least 10 cycles Hydroshear Plus User Manual Page 65 DIG L B Chapter 7 Maintenance 10 Fill and then empty the Hydroshear Plus syringe with distilled or deionized water for at least 10 cycles 11 Leave the input tube open to air And fill the Hydroshear Plus syringe and then eject into an appropriate waste container Repeat procedure 3 times 7 2 3 CLEANING WITH 10 BLEACH 1 Prepare 10 bleach solution by adding part commercial bleach to 9 parts water 2 Fill the Hydroshear Plus syringe with the 10 bleach solution 3 Allow the bleach solution to remain in the pump with the syringe fully lowered for 30 minutes 4 After the 30 minute period remove the reagent tubing from the bleach solution 5 Hill and then empty the Hydroshear Plus syringe once with distilled or deionized water 6 Leaving the inlet tubing open to air fill the Hydroshear Plus syringe and then eject into an appropriate waste container 7 then empty the Hydroshear Plus syringe with distilled or deionized water for at least 10 cycles 8 Leave the input tube open to air And fill the Hydroshear Plus syringe and then eject into an appropriate waste container Repeat procedure 3 times Hydroshear Plus User Manual Page 66 DIG L B Chapter 7 Maintenance 7 3 PERIODIC MAINTENANCE Note It is important that the Hydroshear Plus syringe and plunger are properly aligned for the dev
19. the COM Port text box on the Machine Parameters window as described 1n section 8 3 1 above 3 Ensure that both serial cable connections are securely connected to the Hydroshear Plus and USB connector is securely connected to the controlling computer 8 3 10 PUMP OVERLOAD OR PLUNGER OVERLOAD ERROR This error message appears when the pressure within the syringe becomes so high that the syringe could break This situation is most commonly caused by a clogged shearing assembly Pump overload also can occur if the speed code is too low i e the syringe plunger is moving too fast This is most common when using the small shearing assembly Pump error Initialization error Check For blockages and loose connections before attempting to run protocols or reinitialize Procedure aborted Hydroshear Plus User Manual Page 80 DIG L B Chapter 8 Troubleshooting Pump error Plunger overload The pump must be reinitialized before normal operation can resume Procedure aborted Pump must be reinitialized before protocol can run To resolve these errors follow these steps 1 Reinitialize the pump as described in section 6 1 Explanation of Manual Operation Controls 2 If the error message again appears the shearing assembly is probably clogged Follow the instructions in section 7 4 Unclogging Shearing Orifice Boxes using Sonication to unclog the shearing assembly 3 Check the speed code setting Make
20. to conduct four wash cycles with each of the three wash solutions the total number of wash cycles is twelve Hydroshear Plus User Manual Page 49 L B Chapter 4 Using the Hydroshear Plus Software 4 5 3 CURRENT PUMP VOLUME This box displays the volume of liquid in ul currently in the syringe In the examples shown below the pump volume is 50 ul Unload as 4 5 4 CURRENT VALVE POSITION This box displays the valve that 1s currently open In the example shown below Valve 1 is open Unload Wash 4 6 MACHINE PARAMETERS Several machine parameters that are determined and set at Digilab can be adjusted in the Machine Parameters window New default values can be specified or the saved default values or factory set values can be restored Do not adjust these parameters unless instructed to do so by Digilab If Digilab suggest that these changes be made an explanation of the Machine Parameters can be found in Section 8 3 Software error Messages Hydroshear Plus User Manual Page 50 DIG L B Chapter 5 Shearing DNA Samples Chapter 5 SHEARING DNA SAMPLES 5 1 SETTING SHEARING PARAMETERS Before conducting a shearing run the values for Volume Number of Cycles and Speed Code must be specified in the Hydroshear Plus Main Panel as described in section 4 3 Shearing Parameters It 1s also advisable to set the wash scheme as described section 4 3 7 Setting up a Wash Scheme Read fr
21. usb to 2 rs232 cable windows winS8k amp me mosus A This driver is not digitally signed Tell me why driver signing is important Click Continue Anyway Hardware Installation The software you are installing for this hardware USB to Serial Port Driver has not passed Windows Logo testing to verify its compatibility with Windows me why this testing is important Continuing your installation of this software may impair or destabilize the correct operation of your system either immediately or in the future Microsoft strongly recommends that you stop this installation now and contact the hardware vendor for software that has passed Windows Logo testing Continue Anyway STOP Installation Hydroshear Plus User Manual Page 24 DIG L B Chapter 3 Installing the Hydroshear Plus Click Finish to complete the installation Found New Hardware Wizard Completing the Found New Hardware Wizard The wizard has finished installing the software for Sp USB to Serial Port Driver E Click Finish to close the wizard Finish The USB to serial port converter is now ready to use If there is no communication between the Hydroshear Plus and the computer check that your Ports are properly connected in Device Manager My Computer right click on Properties and select Hardware tab Hydroshear Plus User Manual Page 25 DIG L B Chapter 3 Installing the Hydroshear Plu
22. will automatically insert three pre passes into the shearing run These pre passes will occur before actual shearing cycles and are executed at gradually higher speeds to prepare for the high speed shearing passes Standard shearing assemblies normally should not be operated at a speed code lower than 3 this will produce a pump overload message Small shearing assemblies should not be used at a speed code lower than 6 Remember Lower speed code faster syringe Hydroshear Plus User Manual Page 42 DIGIL B Chapter 4 Using the Hydroshear Plus Software 4 3 7 SETTING UP A WASH SCHEME The Hydroshear Plus should be washed before and after each shearing run to clean any residual sample solution from the machine To edit the parameters used for this wash cycle click on the Edit Wash Scheme button in the Shearing Parameters box 200 00 S Edit Wash Scheme wash Solution I Wash Solution II 1 ash Solution Washing intake spe ing i Washing output speed Cancel 1 Click in the first text box By default this is prefilled with Wash Solution 1 2 In the box to the right of the first wash solution box specify the number of cycles to be performed using the first wash solution This can be accomplished by clicking in the box and typing a number or using the arrows on the left side of the box to adjust the number of cycles 3 Repeat steps and 2 for W
23. DIGILAB Hydroshear Plus DNA Shearing Device User Manual GENERAL INFORMATION Please complete the following section for your records This information should be available to Digilab support staff when contacting us about your Hydroshear Plus Serial Number _ Date Installed Service Contract Type Service Contract Dates CONTACTING DIGILAB Please contact Digilab directly with questions about operating and maintaining the Hydroshear Plus or if the Hydroshear Plus requires servicing To help us assist you please provide the following information The name of your company or institution Your name and contact information The unit serial number located on the back of the instrument A full description of the problem Fax 1 508 893 8011 technicalsupport DigilabGlobal com 100 Locke Drive Marlborough MA 01752 USA DIGILAB LEGAL INFORMATION 2008 Digilab Inc All rights reserved No part of this publication may be reproduced transcribed transmitted or translated into any language in any form and by any means without the written permission of Genomic Instrumentation Services Inc Important notice Digilab believes that the information in this document is accurate The document has been carefully reviewed for technical accuracy In the event that technical or typographical errors exist Digilab reserves the right to make changes to subsequent editions of this document witho
24. If the Volume setting 15 a positive value then liquid will be aspirated from the respective setting If the volume setting 15 negative then the liquid will be dispensed to the respective setting Start button Each time the Start button 1s clicked the syringe plunger and valve position will move according to the settings in the Volume Speed Code and Valve Position boxes If you attempt to enter too large a value the Hydroshear Plus software will warn you as follows The volume of sample is not in the allowable range cannot be outside the range of the syringe volume syringe vol Entry of inappropriate volumes Care should be taken to enter an appropriate value since the syringe pump will have to be reset if the wrong value is entered The following error message would be shown for example if you aspirated 300ul into the syringe and then instead of entering 300 in Volume box to empty the syringe you clicked Start and again tried to aspirate a further 300ul into the syringe thereby exceeding its capacity Pump error Invalid parameter passed to pump Error may have been caused by the pump being told to take in or expel a greater volume than could accommodate Pump may need to reinitialized before attempting further use Procedure aborted To recover from these errors click OK to dismiss this dialog and then reset the syringe pump by clicking on Reinitialize Pump as described above If there is s
25. Microsoft strongly recommends that you stop this installation now and contact the hardware vendor for software that has passed Windows Logo testing Continue amp nyway Hydroshear Plus User Manual Page 20 DIGIL B Chapter 3 Installing the Hydroshear Plus Click Finish to complete the installation Found Mew Hardware Wizard Completing the Found New Hardware Wizard The wizard has finished installing the software for USB Compound Device Click Finish to close the wizard Cancel The Welcome to the Found New Hardware Wizard will appear again to start the installation of the second serial port on the cable Select the radio button No not this time and click Next Found New Hardware Wizard Welcome to the Found New Hardware Wizard Windows will search For current updated software by looking on your computer on the hardware installation or on the Windows Update Web site with your permission Head our privacy policy Can Windows connect to Windows Update to search for software Yes this time only CO now and every time connect a device Click Next to continue Hydroshear Plus User Manual Page 21 DIG L B Chapter 3 Installing the Hydroshear Plus Select the radio button Install the software automatically and click Next Found New Hardware Wizard This wizard helps you install software for USB Compound Device If your hardware came with an
26. OK and the following dialog box appears Bring sample to input tube and click OK 9 Bring sample to the input tube Hydroshear Plus User Manual Page 53 DIG L B Chapter 5 Shearing DNA Samples 10 Remove any extension tubes used to reach wash solutions from both the inlet and outlet tubes Clean the short input tubing by a KimWipe wetted with 70 ethanol or equivalent Place the short input tubing on the right side of the Hydroshear Plus into the tube containing your sample making sure that the input tubing reaches the bottom of the sample We recommend 1 5 ml as a convenient tube size 11 Click OK The specified volume is drawn from the sample tube into Hydroshear Plus input tubing and another dialog appears Remove sample From input tube and click OK 12 Click OK The sample is drawn from the input tubing into the syringe At this point you can discard or set aside the sample tube 5 there an air or bubble in the syringe YES ABORT PROTOCOL 13 Click OK A dialog box appears asking if you have an air bubble in the syringe Is there an air gap or bubble in the syringe YES MO ABORT PROTOCOL 14 If for any reason you wish to cancel the shearing run click on ABORT PROTOCOL Hydroshear Plus User Manual Page 54 DIG L B Chapter 5 Shearing DNA Samples 15 If there is no air bubble in the syringe skip to step 18 16 Ifthere is an air bubble click on YES and the fol
27. TION cccsscccssssccccssccccsssccccssscccssscccessscoees 33 FIGURE 3 4 ATTACHING SHEARING ASSEMBLY TO THE HYDROSHEAR PLUS cccscssscccsssccccsssccccssscoccssccccssscoees 34 FIGURE 3 5 AGAROSE GEL DNA SHEARED AT DIFFERENT SPEED CODES ccccossssscccsssscccsssccccscscocsssccccssscoees 36 FIGURE 7 1 DISMANTLED SHEARING ASSEMBLY cccocsssscccsssccccssscccsssccccsscccsssccccssscccccssccccssccccssscccccssccesescccessscoess 67 FIGURE 7 2 ORIFICE BOX IN FLASK FOR 68 FIGURE 7 52 FLASK IN SONICA TOR 68 FIGURE 7 4 OUTPUT TUBING AND ORIFICE BOX siccacisccacsicccciccciesssececsseasccscusscencceessurecceowssesdesuacacaecccscsceesocossexescens 70 FIGURE 7 5 SAMPLE 71 FIGURE 7 6 LUBRICATING THE DRIVE SCREW 72 FIGURE 9 1 CORRELATION OF SPEED CODE WITH DNA FRAGMENT SIZE ccocssscccssssccccssccccsssccccsssccccssccccssscoees 2 FIGURE 9 2 DETERMINING THE LOWEST STARTING FRAGMENT SIZE ccossssscccsssccccsssccccssccccsssccccssscocsssccccssscoees 83 FIGURE 9 3 DETERMINING THE LOWEST POSSIBLE VOLUME OF STARTING MATERIAL cosssscccssssccccssccccssscoees 84 FIGURE 9 4 SMALLEST SIZE RANGE FOR SMALL SHEARING ASSEMBLY cccsssscccs
28. TS ARE INDEPENDENT OF INITIAL SAMPLE VOLUME Once calibrated the Hydroshear Plus will produce fragments of a given size consistently for a given speed code regardless of the volume of the starting DNA sample Method A 100 ul sample and a 200 ul of lambda DNA were sheared at speed code 5 A 100 ul sample and a 200 ul sample of lambda DNA were sheared at speed code 10 After shearing all four samples were run on an agarose gel Result Each sample resulted in sheared fragments of identical lengths Thus the initial volume of the sample does not affect the post shearing fragment length Lane Volume Speed 100 ul 5 200 ul 5 100 ul 10 200 ul 10 1 kb ladder 1 agarose gel run at 100V for 1 hour All samples are 0 02 ug ul lambda DNA Figure 9 6 Shearing results are independent of starting fragment volume Hydroshear Plus User Manual Page 87 DIG L B Chapter 9 Performance Data 9 7 SHEARING RESULTS ARE INDEPENDENT OF STARTING DNA CONCENTRATION Method Two samples of Lambda DNA with a concentration 0 02 ug ul were sheared at speed code 5 and 10 Two samples of Lambda DNA with a concentration of 0 25 ug ul were sheared at speed code 5 and 10 After shearing all samples of were run on an agarose gel Result Samples of different concentrations resulted in identical fragment lengths when sheared at the same speed code Thus the concentration of the samples did not affect the shearing results for this range of conce
29. TWARE REQUIREMENTS The Hydroshear Plus software and the driver for the interface cable can be installed on any PC fulfilling the following requirements IBM compatible Operating system Microsoft Windows XP One USB port available to connect to the Hydroshear Plus base unit Hydroshear Plus User Manual Page 8 DIG L B Chapter 1 About the Hydroshear Plus 1 5 ELECTRICAL REQUIREMENTS One properly grounded 110 15 amp or 220V 10 amp wall outlet 1 6 ENVIRONMENTAL REQUIREMENTS Pollution Degree 2 Installation Category II Altitude any up to 2000 m Operating Temperature mechanism 59 F 15 C to 104 F 40 C Operating Humidity mechanism 20 95 at 104 F 40 C Hydroshear Plus User Manual Page 9 L B Chapter 2 How the Hydroshear Plus Works Chapter 2 How THE HYDROSHEAR PLUS WORKS The Hydroshear Plus uses a syringe pump and a user adjusted valve to push unsheared DNA samples through a narrow opening The shear forces produced cause the DNA to break into smaller pieces The size of the resulting fragments 1 directly related to the speed at which the DNA moves through the gap After several repeated cycles of shearing the DNA 1 ejected into a collection tube and the Hydroshear Plus is washed in preparation for the next sample A more detailed description follows The innovative design of the Hydroshear Plus appropriates hydrodynamic forces and one way flow though orifice to fragment DNA Thi
30. am Save to File 100 00 _ E NNNM 2 Edit Wash Scheme Restore to Save Default To enter a parameter value change the number in the window next to the parameter either by clicking in the text box and typing a new value or by clicking on the arrows next to the text box to adjust the value In the example shown above a sample with 100 ul volume will be sheared for 20 cycles at a speed code of 12 5 2 PREPARING DNA SAMPLES FOR SHEARING Careful preparation of the sample is imperative A clogged shearing assembly is the most common problem encountered by Hydroshear Plus users and the sample itself usually acts as the clogging material The DNA must be thoroughly resuspended in solution before shearing NOTE Itis recommended to filter the DNA samples prior to shearing to reduce the chance of clogging the shearing assemblies Hydroshear Plus User Manual Page 51 DIG L B Chapter 5 Shearing DNA Samples Ensure that the DNA sample has been resuspended in distilled deionized water ddH20 or an appropriate buffer We recommend either Wash solution 3 or DNase free water NOTE Itis important to have DNA samples free of particles in the DNA which might lead to clogging 5 3 SHEARING DNA SAMPLES 1 Click the START button located on the Hydroshear Plus Main Panel A dialog box appears asking if you want to perform a wash cycle Are vau ready bo start washing 2 Click YES unless the Hydrosh
31. ample in the syringe and the default position of the valve 15 Input place the container at input to collect the sample Hydroshear Plus User Manual Page 61 DIGILAB Chapter 6 Manual Operation 6 2 MANUAL ASPIRATION INTO THE HYDROSHEAR PLUS SYRINGE The following procedure should only be used when the syringe is empty To draw a volume of fluid or air into the pump follow these steps 1 Locate the Status box on right hand side of Hydroshear Plus Main Window 2 Verify that 0 appears in the Current pump volume text box to indicate that there is no fluid in the Hydroshear Plus syringe 3 Click on the Manual Operation button to open the Manual Operation window 4 Valve Position of Input is selected the aspiration will be from the input tube If it is desired to aspirate from one of the Wash tubes then the appropriate Wash tube must be selected E Manual Operation Reinitialize Pump mo Start 5 Inthe Volume box enter a positive value for the volume you wish to aspirate into the syringe For example to aspirate 350ul enter 350 in the Volume box 6 Inthe Speed Code box specify the speed code at which you wish the syringe pump to move Remember the higher the speed code the slower the syringe pump will move For manual operation 10 1s a reasonable speed code Choose 20 if the shearing assembly is clogged or if you want a slow syringe movement
32. anged any changes made here will not be applied to the shearing parameters The values stored in the saved file will be applied Hydroshear Plus User Manual Page 46 DIGILAB Chapter 4 Using the Hydroshear Plus Software 4 3 10 SAVING NEW DEFAULT SHEARING PARAMETER VALUES If you use one set of shearing and washing parameters routinely you can save these as the default settings In this way they will be preset whenever the Hydroshear Plus software 1 started To change the default settings in this way enter the desired values for shearing and washing as described in section 4 3 4 Editing Basic Shearing Parameters and section 4 3 7 Setting up a Wash Scheme and then click on the Save as Default button in the Shearing Parameters box of the Hydroshear Plus Main Panel I Restore to Default Save Default A dialog box will appear asking you to confirm this action If you are sure you want to replace the current default settings click Replace Replace existing C Program FilesdHvdrashearYuserDefaulEs Cancel These saved values will now be used as the starting values for the Hydroshear Plus software Remember that these saved default values include those for shearing and for the wash scheme Clicking the Save as Default button will save both the currently entered shearing parameters as well as the currently entered wash scheme values even though the wash scheme 1 not visible Make sure that
33. ash Solution 2 and Wash Solution 3 Hydroshear Plus User Manual Page 43 DIGIL B 4 A typical wash scheme would be 4 cycles of each wash as shown above Chapter 4 Using the Hydroshear Plus Software 5 Enter the speeds you wish to use during the wash and Click OK to exit this window and save these wash scheme steps NOTE For the standard and large shearing assemblies the default of 6 for the Intake Speed and 12 for the Output speed are acceptable For the small shearing assembly it is recommended to change the Intake speed to 10 and Output speed to 14 Hydroshear Plus User Manual Page 44 DIGIL B Chapter 4 Using the Hydroshear Plus Software 4 3 8 SAVING SHEARING PARAMETERS VALUES TO A FILE Custom shearing parameters can be saved to a file allowing for easy recall and entry when conducting future shearing runs In this way if you have several samples that are sheared frequently the shearing parameters for each of these samples can be stored for easy access This file 1 user named and will be stored in the user specified location To save shearing parameters to a file follow these steps 1 After entering values for each basic shearing parameter and for the wash scheme as described above click the Save to File button Read fram File Save to 2 The save file dialog box appears Choose file to write Save Co 209 t Hydroshearz09 exe Ei iw
34. both are set correctly before saving new default conditions Hydroshear Plus User Manual Page 47 L B Chapter 4 Using the Hydroshear Plus Software 4 3 11 RESTORING SHEARING PARAMETER DEFAULT VALUES If after changing the shearing parameters i e shearing parameters and or wash settings you should decide that one or both were not appropriate for the shearing run you wish to use it 15 possible to revert the default values To do this click on the Restore to Defaults button located at the bottom left corner of the Shearing Parameters box on the Hydroshear Plus Main Panel This will change all of the currently set parameters both shearing and washing to the last saved default values and display those in the appropriate text boxes L _ Restore to Defaults Save 4s Default 4 4 OPERATION CONTROLS Five buttons are located in the Operation Controls box on the Hydroshear Plus Main Panel Click the START button to start a shearing run after setting the appropriate values Click the STOP button to stop a shearing run already in progress The machine will stop after the current pump pass is complete Click the PAUSE button to pause the current shearing run The machine will complete its current shearing pass and then pause While paused click this button again to resume the shearing run Do not repeatedly click this button to speed up the software and or machine 5 response each click is registered an
35. cts and demonstration remanufactured or refurbished Products will be free from defects in material and workmanship for a period of ninety 90 days after shipment Seller s obligation under these warranties will be limited to repair or replacement of any defective Products Seller shall have the sold authority to determine the type and means or repair or replacement in the event of a claim Seller shall have the option of requiring the return of defective products transportation prepaid to determine the claim Seller shall not be responsible for repairs made by others There are also offers Depot Only Extended Warranty Programs available Please contact Digilab for more information Hydroshear Plus User Manual Page 7 DIG L B Chapter 1 About the Hydroshear Plus 1 2 COMPONENTS In addition to the base unit and software CD each Hydroshear Plus comes complete with the following 1 Power cord 2 USB to 2x RS 232 serial cable includes CD with drivers for cable 3 500 ul syringe 4 Four shearing assemblies with tubing attached 2 standard I large and I small 5 Tool kit 6 Hydroshear Plus Software CD 7 Hydroshear Wash Solution Kit box with 3 wash solutions 8 Empty waste bottle 9 Laptop with preloaded software optional 1 3 TECHNICAL SPECIFICATIONS Fuse Current Rating 3 Amps Fuse Voltage Rating 250V Voltage 100 250 VAC Frequency Range 47 100 Hz Installation Over voltage Category 1 4 COMPUTER SOF
36. d acted upon Thus repeated clicking will start a cycle of pause unpause pause unpause and so on Click the MANUAL OPERATION button to display the Manual Operation window where you can conduct individual shearing steps See Chapter 6 Manual Operation Click the WASH button to wash the shearing assembly The wash parameters can be changed Edit Wash Scheme Section 4 3 7 Hydroshear Plus User Manual Page 48 DIGILAB Chapter 4 Using the Hydroshear Plus Software 4 5 STATUS The Status box provides information on four items Current Step Run Status Current Pump Volume and Current Valve Position and is useful when diagnosing and resolving troubleshooting issues See Chapter 8 Troubleshooting page 72 4 5 1 CURRENT STEP This box displays list of each step in the current shearing run When a shearing run is in progress the current step 1 highlighted 4 5 2 RUN STATUS Under Completed on the right side of the Status box the progress of the current step 15 displayed Progress is displayed in the following format lt number of cycles completed gt of lt total number of cycles in step gt In the example below 20 of 20 shearing pass steps has been completed Wash Load Purge Air tun Unload Wash Note that when viewing the progress of a wash step the total number of cycles displayed 15 the total using all three wash solutions For example if you specified in your wash scheme that you would like
37. e These values can be adjusted for each run If you use the same parameters frequently you can save these as the default values so that they are already preset when the software starts up NOTE the actual shearing procedure has been started the shearing parameters cannot be changed 4 3 1 VOLUME This is the volume of the sample in microliters u you are preparing to shear This value can range from the void volume to the full syringe volume 500 ul However we have found that shearing will be incomplete unless the volume is at least 40 ul The Hydroshear Plus default is set for 200 ul Hydroshear Plus User Manual Page 40 DIG L B Chapter 4 Using the Hydroshear Plus Software 4 3 2 NUMBER OF CYCLES This number refers to the number of shearing passes the Hydroshear Plus will perform on the sample The default number of cycles 1 20 since after the twentieth cycle additional shearing passes are unlikely to shear the sample any further 4 3 3 SPEED CODE The speed code correlates with the speed of the syringe pump when performing the shearing passes The optimal value depends on both the individual orifice size of the shearing assembly there are slight variations in size among orifices and the fragment size desired The speed code can range from 0 to 40 with an increasing speed code value corresponding with a decreased pump plunger speed A speed code of 0 not recommended for actual shearing runs results in t
38. e 1 open Please follow the instructions below before using a new Hydroshear Plus syringe to ensure that the syringe and plunger are aligned correctly with the valve assembly l Lower the plunger mount on the pump Since the plunger mount cannot be lowered manually with the Hydroshear Plus turned off turn on the Hydroshear Plus using the switch on the rear of the unit 2 Start the Hydroshear Plus software on the attached PC The main Hydroshear Plus control panel 15 as follows 1 HydroShear Main Panel 3 main Hydroshear Plus control panel click on MANUAL OPERATION to access manual syringe controls Hydroshear Plus User Manual Page 28 DIG L B Chapter 3 Installing the Hydroshear Plus MANUAL OPERATION 4 In the Manual Operation window enter 500 in the Volume box and 10 in the Speed Code box and then press START 8 Manual Operation 5 the syringe motor plunger has stopped moving exit the Hydroshear Plus software turn the instrument off and disconnect the power cable from the wall outlet 6 Push the plunger into the syringe so that it will fit between the adaptor at the top and the plunger mount at the bottom 7 Remove the syringe mount screw that is connected to the pump 8 Screw the syringe counterclockwise into the syringe mount Hydroshear Plus User Manual Page 29 DIG L B Chapter 3 Installing the Hydroshear Plus Syringe Mount Tip of syringe screws
39. e assemblies proceed as instructed by the software prompts as outlined in section 5 3 1 Sample Ejection from Shearing Assemblies 5 3 1 SAMPLE EJECTION FROM SHEARING ASSEMBLIES 1 When Shearing cycles complete message appears click OK and you will be asked to confirm sample ejection from the Hydroshear Plus 2 sure there is a container at the output tube to collect the sample and click YES The following dialogue box will then appear Hydroshear Plus User Manual Page 56 DIG L B Chapter 5 Shearing DNA Samples Are vou ready to shart washing 3 As stated previously you must wash the device after each sample to ensure that there is no cross contamination between different samples Click on YES to run the wash scheme defined for the shearing run The steps for washing are described below in section 5 4 Washing the Hydroshear Plus 5 4 WASHING THE HYDROSHEAR PLUS Before and after the Hydroshear Plus performs a shearing run you will be asked if you wish to wash the device There is a Wash button in the Operations Control section of the software and if you click on Wash software will prompt you through an entire wash cycle Do vau want to wash the device before BEFORE SHEARING starting the shearing process AFTER SHEARING Hydroshear Plus User Manual Page 57 DIG L B Chapter 5 Shearing DNA Samples 5 4 1 AUTOMATED WASHING OF THE SHEARING ASSEMBLIES 1 Place t
40. e menu and click on the CD drive where the Hydroshear Cable CD was loaded You must use the same USB port on the computer each time the USB to 2X Serial Connector cable is connected otherwise the converter cable drivers will need to be re installed There is a selection of cable types listed Double click on the USB to 2X RS232 icon as circled in red below Driver D SEE gt File Edit View Favorites Tools Help Back 2 27 E Search ie Folders Address D v Go Folders x Files Currently on the CD 8 03 drivers a m x 2s 2 exact 0 Bp Bp P B f48ad320f524827469bd79b72F 2 i386 USB2 0 EASY USB 2 0 Easy USB 2 0 USB 2 0 TO USB 2 0 TO USB Link Cable B l TRANSFE Link Met Cable Network Cable R5232 Cable R5232 C C3 MyIDM gt gt ET 23 Program Files CJ CJ Qoobox USB USB R5232 USB 5232 C3 SwDist d R5232 Cable R542 Cable Parallel Prin Cable Converter 3 temp WINDOWS 7 eu autorun autorun C3 USB2 0 EASY TRANSFER LINK CABLE FOF O USB 2 0 Easy Link Net Cable USB 2 0 Network Cable C3 USB 2 0 TO R5232 Cable USB 2 0 R5232 Converter USB Link Cable USB Network Cable C3 USB TO 2 x R5232 Cable O Linux B mac O manual 8 windows amp USB TO 2 x R5422 Cable 22 USB TO Parallel Printer Cable lt The following screen will appear In order
41. ear Plus has just been cleaned This may be the case if you are conducting multiple shearing runs Always clean the Hydroshear Plus before the first run of the day even if it was cleaned after the last run of the previous day 3 If you clicked YES the specified wash scheme is performed For a step by step explanation of the on screen prompts and corresponding user actions involved in this process see Section 5 4 Washing the Hydroshear Plus Upon completion of the prewash cycles a dialog box appears informing you that the Hydroshear Plus prewash is complete Click OK to continue Washing complete Hydroshear Plus User Manual Page 52 DIG L B Chapter 5 Shearing DNA Samples 4 Another dialog box appears asking if you want to continue the shearing process by loading the sample 5 sample is already in the syringe you should click NO In most cases you will not have loaded the sample yet and should click YES If for any reason you do not wish to continue at this point for example if you discovered that your sample was not ready or you want to change the shearing parameters you can click on ABORT PROTOCOL to exit the shearing run Proceed with loading the sample YES NO ABORT PROTOCOL 6 After clicking YES a dialog box appears 7 Make sure you have your sample prepared such that it is thoroughly in solution as described above in Section 5 2 Preparing DNA Samples for Shearing 8 Click
42. empting to attempting to set slope set slope To correct this problem dismiss these dialog boxes and then click on the Edit Machine Parameters button Change the Port Number to the correct value and click on Save as Default Hydroshear Plus User Manual Page 78 DIGILAB Chapter 8 Troubleshooting 8 3 8 SETTING NEW DEFAULT MACHINE PARAMETER VALUES To set new default machine parameters either click in the text box and type in the desired value or click the arrows next to the text box to adjust to the desired value Then click the Save as Default button 8 Machine Parameters A dialog appears asking for confirmation If you are sure you want to do this click Replace you are not sure about this course of action click Cancel The new values are saved and will be used as the default values for every shearing run Note that as stated previously there is usually no need to change these settings Replace existing C Program Files Senomics Hydroshearz09 i machineDefaults Hydroshear Plus User Manual Page 79 DIG L B Chapter 8 Troubleshooting 8 3 9 READ TIMEOUT ERROR This error message appears when the Hydroshear Plus and control computer are not communicating Read timeout while attempting sek slope To resolve this error follow these steps 1 sure Hydroshear Plus is plugged in and turned on 2 Check to see that the correct CPU serial port number is entered in
43. ese udo usa 50 4 6 MACHIN PABAMETEBS 50 5 SHEARING DNA SAMPLES 5 1 SETTING SHEARING PARAMETERS ii es so cerae oo n sue va Eno sa ni ER 51 5 2 PREPARING DNA SAMPLES FOR 51 5 3 SHEARING DNA SAMPLES C 52 5 3 1 SAMPLE EJECTION FROM STANDARD AND LARGE SHEARING ASSEMBLIES 56 5 4 WASHING THE HYDROSHEAR PLUS eseesseessesssesssossesssossesssesseossesssossossessseossesseossesssessesssesssosseo 57 5 4 1 AUTOMATED WASHING OF THE SHEARING ASSEMBLIES essssssesssossesssossesoscssssosesssossesssssoe 58 5 4 2 RECOMMENDED WASH SOLUTIONS sseosseosssesssesssccsssosssesosessssesssesssossssesssesoseosssesosesossssssessse 59 CHAPTER 6 MANUAL OPERATION 6 1 EXPLANATION OF MANUAL OPERATION CONTROLS cccccccscccscssscccccccscssccccccccceccccsccsscccscccsces 60 6 2 MANUAL ASPIRATION INTO THE HYDROSHEAR PLUS SYRINGE sssesesescscecesesescsososoeoesesoooe 62 6 3 MANUAL EJECTION FROM THE HYDROSHEAR PLUS SYRINGE 63 CHAPTER7 MAINTENANCE 7 1 GENERAL MAINTENANCE GUIDELINES cccsscccsssccccsccccscsccccccccccccccccccccssccccscccccscccccscsccescsceeees 64 1 2 PERIODIC EO SLT Cr TaS 64
44. essssesoe 81 CHAPTER9 PERFORMANCE DATA 9 1 RELATIONSHIP OF FRAGMENT SIZE TO PUMP SPEED ccccccssccsssccsccccsccccscccccccccsccccccccscceescees 82 9 2 SMALLEST STARTING FRAGMENT SIZE THAT CAN BE SHEARED 83 9 3 SMALLEST STARTING SAMPLE VOLUME THAT CAN BE SHEARED 84 9 4 SMALLEST FRAGMENTS THE HYDROSHEAR PLUS CAN CONSISTENTLY PRODUCE 85 9 5 INITIAL FRAGMENT LENGTH DOES NOT AFFECT SHEARING PERFORMANCE ccccccscccssceees 86 9 6 SHEARING RESULTS INDEPENDENT OF INITIAL SAMPLE VOLUME ccccsccccscccssccccscccscees 87 9 7 SHEARING RESULTS INDEPENDENT OF STARTING DNA 88 DIGILAB LIST OF FIGURES FIGURE 2 1 HOW THE HYDROSHEAR PLUS WORKS ccsccsscsssssessccccsscssocccscsscccssssscccccsscesscccsscescocssscessoosescessoesess 10 FIGURE 2 2 HYDROSHEAR PLUS VALVE POSITIONS 12 FIGURE 3 1 REAR VIEW OF HYDROSHEAR PLUS 26 FIGURE 3 2 SHEARING ASSEMBLY COMPONENTS scccccccssssssccccsssscccccssssccccssssccccessssscccccssssccccssssssccessssscccsesssscossees 31 FIGURE 3 3 DETERMINING CORRECT SHEARING ASSEMBLY ORIENTA
45. et to the Factory defaults IF wou don t wish to use the Factory settings vau should specify other values before beginning shearing protocol 4 Click OK to dismiss the dialog box Hydroshear Plus User Manual Page 37 L B Chapter 4 Using the Hydroshear Plus Software Enter Port Number work offline 5 Select the COM ports for the pump and valve The driver for the serial USB cable adds two COM ports to the computer There 1 no way to know which COM port to select for the pump and valve initially however if your selection 1s incorrect there will be a prompt stating that they are not found If this happens switch the COM port selections between the pump and valve 6 Change the automated valve selection switch to YES If NO is chosen Hydroshear Plus will operate similar to the Hydroshear and will use only Input and Output ports and will 1gnore the wash ports In this mode you will be prompted to change the wash solutions 7 Click Connect 8 At this point the syringe plunger and valve of the Hydroshear Plus should reinitialize and the Hydroshear Plus Main Panel becomes available 9 feither the valve or the pump is recognized but not both and you are certain that you have assigned the correct port click Connect again Hydroshear Plus User Manual Page 38 L B Chapter 4 Using the Hydroshear Plus Software 4 2 OVERVIEW OF THE MAIN CONTROL PANEL The Hydroshear Plus Mai
46. furnished to Customer under a nonexclusive license for use only on the single unit of Product for which the Software is provided Such Software may not be used on or with any other Products or for activities outside the scope of this agreement without the prior written permission of the Company Customer acknowledges that the Software constitutes valuable proprietary confidential and trade secret information of the Company or of third party suppliers of Software as the case may be Such Software may not be copied in whole or in part or sublicensed provided transferred or otherwise disclosed or made available to third parties without the express prior written permissions of the Company or such third party Software supplier as the case may be Customer will notify the Company promptly of any unauthorized use possession knowledge of Company or such third party Software supplier as the case may be If requested by the Company Customer will execute and deliver a standard form or license agreement issued by the Company supplier with respect to any Software provided hereunder that is owned and licensed by such supplier The software is protected by United States Copyright Law and International Treaty provisions Under the copyright laws this Software may not be reproduced or transmitted in any form electronic or mechanical including photocopying recording storing in an information retrieval system or translating in whole or in part without the pri
47. gth were sheared using a small shearing assembly at a speed code of 3 The resulting fragments were then run on an agarose gel Result All three samples results in a fragment size range of 750 bp to 1 5 kb with the majority of fragments centered around 1 kb 1 agarose gel run at 105V for 1 hour All 50 pisamples were taken from the same stock of 20ng ul lambda Figure 9 4 Smallest size range for small shearing assembly Hydroshear Plus User Manual Page 85 DIGIL B Chapter 9 Performance Data 9 5 INITIAL FRAGMENT LENGTH DOES NOT AFFECT SHEARING PERFORMANCE Once calibrated the Hydroshear Plus will produce fragments of a given size consistently for a given speed code regardless of the size of the starting DNA sample Method Three samples with sizes of 10 48 5 and 180kb respectively were sheared at speed code 5 The resulting fragments were then run on an agarose gel Result Each sample resulted in sheared fragments of identical lengths Thus the initial fragment length of the sample does not affect the post shearing fragment length Starting Size 3000 bp 10 kb 48 5 kb 4 180 kb 1 5 Ikb ladder 2000 bp 1000 bp agarose gel run at 105V for 1 hour All samples sheared at pump speed 5 Figure 9 5 Shearing results are independent of starting fragment length Hydroshear Plus User Manual Page 86 DIGILAB Chapter 9 Performance Data 9 6 SHEARING RESUL
48. gure 7 2 Orifice box in flask for sonication 7 the sonicator with enough water so that the level will be higher than the level of fluid in the flask when the beaker 15 placed in the sonicator If your sonicator has a minimum fill level be sure to fill up to that level 6 Place flask with the shearing orifice box into the sonicator If necessary use a weight to keep the container submerged Figure 7 3 Flask in sonicator 9 Visually inspect the shearing devices to determine if any air bubbles are present in the openings of the orifice box If air bubbles are present keep the orifice box submerged and use a micropipette with a sterile tip to either dislodge or aspirate the bubble Take care not to contact the ruby in the center of the orifice box 10 Sonicate the submerged orifice box for 10 15 minutes 11 After sonication pour the SDS solution out of the flask 12 Leave the orifice box in the flask and rinse with ultrapure distilled water Hydroshear Plus User Manual Page 68 DIGIL 13 14 15 16 17 18 19 20 2L 22 Chapter 7 Maintenance Add fresh ultrapure water to the flask with orifice box and inspect for air bubbles Sonicate for 10 15 minutes Pour the water out of the flask leaving the orifice boxes inside Pour isopropyl alcohol into the flask to cover the orifice box Place the flask into the sonicator inspect for air bubbles as before and sonicate for 10 15 minu
49. he Hydroshear Plus output tubing into a waste container and click YES 2 Hydroshear will then complete a wash procedure When complete in order to clear the input tube the following dialog box will appear Remove any liquids From Ehe opening of the input tube and click In order to ensure input tube is clear the same procedure will be repeated Remove any liquids From Ehe opening of the input tube and click To wash the input tube select OK To wash input port attach extension tube and click OK Hydroshear Plus User Manual Page 58 DIG L B Chapter 5 Shearing DNA Samples To clear all liquids from the shearing port tube select OK Remove any liquids From Ehe opening of shearing port tube and click OK Click OK when the washing is complete 5 4 2 RECOMMENDED WASH SOLUTIONS In order to prevent clogging of the shearing assembly all wash solutions should be filter sterilized using a filter of 0 2 microns or less Wash solutions should be made up using distilled and deionized water never use tap water See Page 27 Section 3 4 for details on wash solutions Hydroshear Plus User Manual Page 59 DIGILAB Chapter 6 Manual Operation Chapter 6 MANUAL OPERATION The Hydroshear Plus syringe plunger can be manually controlled using the Manual Operation window which allows control over three functions Aspirating fluid into the syringe Expelling fluid from the syringe
50. he default directory which is C Program Chapter 3 Installing the Hydroshear Plus File Hydroshear 4 Select the radio button I accept the License Agreement and click Next Hydroshear Plus User Manual HydroShear Destination Directory Select the primary installation directory All software will be installed in the following locations To install software into different location s click the Browse button and select another directory Directory for HydroShear C Program Files HydroShear s Directory for National Instruments products C Program Files N ational Instruments E HydroShear License Agreement You must accept the license s displayed below to proceed NATIONAL INSTRUMENTS SOFTWARE LICENSE AGREEMENT INSTALLATION NOTICE THIS IS CONTRACT BEFORE YOU DOWNLOAD THE SOFTWARE AND OR COMPLETE THE INSTALLATION PROCESS CAREFULLY READ THIS AGREEMENT BY DOWNLOADING THE SOFTWARE AND OR CLICKING THE APPLICABLE BUTTON TO COMPLETE THE INSTALLATION PROCESS YOU CONSENT TO THE TERMS OF THIS AGREEMENT AND YOU AGREE TO BE BOUND BY THIS AGREEMENT IF YOU DO NOT WISH BECOME A PARTY TO THIS AGREEMENT AND BE BOUND BY ALL OF ITS TERMS AND CONDITIONS CLICK THE APPROPRIATE BUTTON TO CANCEL THE INSTALLATION PROCESS DO NOT INSTALL OR USE THE SOFTWARE AND RETURN THE SOFTWARE WITHIN THIRTY 30 DAYS OF RECEIPT OF THE SOFTWARE INCLUDING ALL ACCOMPANYING WRITTEN MATERIALS ALONG WITH THEIR CONTAINERS
51. he fastest speed 1 2 seconds per stroke while a speed code of 40 results in the slowest speed 600 seconds per stroke The default value on the Hydroshear Plus 1s 8 4 3 4 EDITING BASIC SHEARING PARAMETERS To edit a parameter value locate the text box next to the parameter name e g Volume Then either click in the text box and type in the desired value or click the arrows next to the text box to select the desired value Should you edit the shearing parameters and wish to restore the default values click the Restore to Defaults button located at the bottom left corner of the Shearing Parameters box This will replace the numbers in the text boxes with the last set of values saved as default values Note that restoring default shearing parameters will also restore default washing parameters too Washing parameters are described below in section 4 3 7 4 3 5 FINDING THE MAXIMUM SPEED CODE In addition to calibrating each new shearing assembly to determine which fragment sizes are generated by each speed code new small assemblies should also be tested to establish the maximum acceptable speed code for shearing Both of these calibrations should be performed on the small assembly before any shearing runs are attempted Hydroshear Plus User Manual Page 41 DIGIL B Chapter 4 Using the Hydroshear Plus Software To find the fastest allowable speed shearing runs should be performed without any DNA using distilled water o
52. hearing process or to the sheared DNA However solutions used in the washing process could damage or destroy DNA Therefore 1f the syringe was inadequately purged after washing leftover wash solution could have damaged or destroyed the DNA being sheared Hydroshear Plus User Manual Page 75 DIGILAB Chapter 8 Troubleshooting 8 3 SOFTWARE ERROR MESSAGES 8 3 1 ERROR 37 SERIAL PORT This message will appear if the wrong CPU serial port number 15 entered as the Port Number in the Machine Parameters window Error 37 occurred ak Serial Pork Inik Possible reasons LabVIEW Device not Found To resolve this error check to see that the correct serial port number is entered in the Port Number text box on the Machine Parameters window For almost all users this will be set to zero 8 3 2 MACHINE PARAMETERS If you have been instructed by Digilab to change any of the Machine Parameters the following describes each of the settings Machine Parameters Hydroshear Plus User Manual Page 76 DIGIL B Chapter 8 Troubleshooting 8 3 3 SYRINGE VOLUME This value 15 the volume of the syringe in ul that you have installed on your Hydroshear Plus The volume of the standard syringe supplied with the Hydroshear Plus 15 500u1 8 3 4 VOID VOLUME This value 15 the volume of fluid that must remain in the orifice during shearing The accuracy of this value 1s imperative for proper shearing If the vo
53. ice to work properly Visually check the alignment of the syringe after conducting any maintenance procedures If the syringe does not appear to be parallel to the Hydroshear Plus case one of the components may be misaligned If this 1s the case follow the instructions in Section 3 5 installation to realign the syringe and plunger 7 4 UNCLOGGING SHEARING ORIFICE BOXES USING SONICATION During the course of using the Hydroshear Plus to shear DNA shearing assemblies can sometimes become clogged Because the Hydroshear Plus works using a restricted flow orifice it 1 necessary that fluids are able to flow through the shearing assembly Sonication can be used to unclog shearing orifices as described below Note that these instructions show a single orifice box but it 1s possible to sonicate multiple boxes simultaneously 1 Loosen the shearing assembly from Hydroshear Plus output port using the 1 4 hex wrench 2 Remove the shearing assembly by unscrewing it the from the output port 3 Unscrew the nuts connecting the tubing to the orifice box 4 01234 1 STANDARD Figure 7 1 Dismantled shearing assembly 4 Prepare a 1 solution of SDS sodium dodecyl sulphate in ultrapure water 5 Place the orifice box into a glass flask 6 Pour the SDS solution into a flask so that the orifice box is completely submerged Hydroshear Plus User Manual Page 67 DIGIL Chapter 7 Maintenance Fi
54. il the pieces are too short for the drag forces to break the molecular bonds The final fragment size i e the length at which the shearing force 15 too weak to break the bonds 15 determined by the flow rate of the fluid and the size of the shearing assembly orifice Hydroshear Plus User Manual Page 10 L B Chapter 2 How the Hydroshear Plus Works 2 1 SHEARING ASSEMBLY In order to accomplish accurate reproducible shearing the Hydroshear Plus utilizes a precision drilled ruby as its shearing orifice The ruby is mounted inside the orifice box which is connected by tubing to the valve system 2 2 PUMP SYSTEM The Hydroshear Plus employs a syringe pump system Air and fluid flow is pressure controlled by the movement of the plunger inside the glass syringe The pump will stall if this pressure 1 too high It 1s also likely that leaks will occur in the Hydroshear Plus tubing and or valves if operated at higher than normal pressures The most common cause of increased pressure is a blocked shearing assembly as described in section 8 3 10 Pump Overload or Plunger Overload Hydroshear Plus User Manual Page 11 L B Chapter 2 How the Hydroshear Plus Works 2 3 VALVE SYSTEM The Hydroshear Plus utilizes a single seven port valve The default positions for the valve are as labeled below These positions can be redefined in the Hydroshear Plus software Port 1 Port 2 Sample Input To shearing assembly a
55. lowing dialog box will appear Where is the bubble Middle of syrynge Depending upon which answer is selected the Hydroshear Plus will go through a sequence of plunger movements and valve actuations in order to clear the bubble When complete a dialog box will appear again and inquire about bubbles 5 there air gap or bubble in the syringe YES ABORT PROTOCOL Step 17 may have to be repeated a few times in order to clear the bubbles 17 The Is there an air gap or bubble in the syringe message box will appear again Hopefully at this point you will be able to click on NO as the answer to this question However if there 15 still a bubble click on YES and repeat step 17 18 Irrespective of how you got to this point click on NO in the Is there an air gap or bubble dialog box and the Hydroshear Plus conducts the specified number of shearing passes The number of passes completed 1 updated in the status area of the Hydroshear Plus Main Panel Upon completion of the shearing cycles a dialog box appears informing the user that the shearing process has ended Hydroshear Plus User Manual Page 55 DIG L B Chapter 5 Shearing DNA Samples IMPORTANT We have discovered that in some cases the speed code used for sample ejection is too low for the small shearing assembly and can cause Plunger Overload errors or sample leakage from the shearing assembly or the connecting tubing For standard and larg
56. lume entered is too large some DNA will be left unsheared because the fluid will not be retracted far enough into the syringe If the volume entered is too small fluid will be retracted too far past the orifice creating a jet effect DNA will move through the orifice so quickly that the resultant fragments will be shorter than the speed code would indicate For the standard 500 ul syringe the void volume should be 15 ul 8 3 5 RETRACTION SPEED This value refers to the speed of the pump during the retraction pass and corresponds with the number of seconds required to complete one pump pass The retraction speed must be kept slow relative to the speed code used to shear samples This prevents the creation of air bubbles in the sample and prevents shearing from occurring during the retraction pass For the small shearing assembly we recommend a starting retraction speed code of 32 This should ensure that all of the sample will pass back through the orifice during each cycle It should also minimize the formation of air bubbles in the sample Note that this speed code of 32 1s the suggested starting value and this may need to be adjusted for different individual shearing assemblies and also on the sample being sheared It 1s recommended that users observe the sample during the shearing run If the entire sample does not pass back into the assembly or if there 1s excessive bubble formation then the speed code should be increased i e slowing the s
57. mple is retracted from the output tubing at a sporadic rate The sample continues to move after the syringe has stopped To rectify this problem 1 Click the STOP button to stop the shearing run See section 4 4 Operation Controls 2 Use the Manual Operation window to eject the DNA sample See section 6 3 Manual Ejection from the Hydroshear Plus Syringe Store the DNA sample since you will want to resume the shearing run later 3 Unclog the shearing assembly See section 7 4 Unclogging Shearing Orifice Boxes using Sonication Hydroshear Plus User Manual Page 73 DIGIL B Chapter 8 Troubleshooting 8 1 3 DECREASING SAMPLE VOLUME Most likely a fitting valve or the syringe 1 leaking Tighten all the tubing connections and screws If the problem persists contact Digilab for assistance 8 1 4 INCREASING SAMPLE VOLUME This may occur on a regular basis because of minimal liquid leftovers remaining within the output tubing after the washing procedure is completed If dilution of your sample is a problem you may concentrate the sample by precipitating the DNA fragments with ethanol or iso propanol according to the procedures of your choice The use of membrane based concentrators is not recommended 8 2 SAMPLE AND FRAGMENT PROBLEMS 8 2 1 LOSS OF DNA Losing DNA 15 most likely a result of inadequate purging of the syringe after washing If the syringe 1s not properly purged after washing left
58. n Panel is the Hydroshear Plus s control and feedback center The Hydroshear Plus Main Panel is divided into three boxed areas titled Shearing Parameters Operation Controls and Status These are summarized as follows with more detailed descriptions in the following sections Shearing Parameters This box 1 used to customize parameters before a shearing run save customized shearing parameters to a file instruct the software to use parameters saved to a file or set new default parameters Operation Controls This box includes the buttons used to run the Hydroshear Plus and a button used to navigate to the Manual Operation window See 6 Manual Operation page 57 Status This box displays the status of a current shearing run or wash as well as the current pump volume and valve position Hydroshear Plus User Manual Page 39 Chapter 4 Using Hydroshear Plus Software HydroShear shearing Parameters Read fram File Save to File Operation Controls MANUAL OPERATION WASH Status volume Current Step Number af cycles 20 Wash Completed i Load Wash 0 of 0 Speed Code Purge Air Run Current pump volume Unload Wash 50 Current valve position 1 4 3 SHEARING PARAMETERS Three basic shearing parameters are referenced by the Hydroshear Plus software Volume Number of cycles and Speed Code The default value for each is displayed upon starting the softwar
59. nd Sample Output Port 6 Port 3 Wash Solution 3 Closed Port 7 To syringe pump Port 5 Wash Solution 2 Port 4 Wash Solution 1 Figure 2 2 Hydroshear Plus Valve Positions Hydroshear Plus User Manual Page 12 DIG L B Chapter 3 Installing the Hydroshear Plus Chapter 3 INSTALLING THE HYDROSHEAR PLUS 3 1 FINDING A LOCATION FOR THE HYDROSHEAR PLUS Select a location for the Hydroshear Plus that meets the following guidelines properly grounded 110V 15amp wall outlet should be within reach of the Hydroshear Plus power cord The Hydroshear Plus and the controlling CPU monitor should be placed in full view of each other Choose a location away from any vents that could expel particulate material on machine and contaminate the sample Choose a location such that the Hydroshear Plus can be placed on a flat level surface Ensure that the location satisfies the Hydroshear Plus s environmental requirements NOTE It may be advisable to use an APC Power Supply to prevent loss of sample in the case of a power outage 3 2 SOFTWARE INSTALLATION 3 2 1 HYDROSHEAR PLUS 1 Insert the Hydroshear Plus installation CD into the computer 2 file setup exe from the installation disk by double clicking on it Hydroshear Plus User Manual Page 13 DIGILAB 3 dialog will appear asking which directory the software should be installed into There is no need to change from t
60. ntrations Lane Concentration Speed 0 02 ug ul 5 0 25 ug ul 5 0 02 ug ul 0 25 ug ul kb ladder Mbp Ld agarose gel run at ISV for 1 haour samples are Lambada ONA Loaded 02 pe of each sample Figure 9 7 Shearing results are independent of starting fragment concentration Hydroshear Plus User Manual Page 88 DIGILAB 100 Locke Drive Marlborough MA 01752 USA 508 893 3130 www digilabglobal com
61. olheane for LISB 0 5 Driver Lick Finish bo close tha vend The Found New Hardware Wizard will appear a third time Select the radio button No not this time and click Next Found New Hardware Wizard Welcome to the Found New Hardware Wizard Windows wil search for current and updated software by looking on your computes on the hardware installabon on the Windows Update Web site with your permission Read our pavacy policy Can Windows connect to Windows Update to search for soltware O Yes this time ordy Q Yes now and every tme connect a device No not this tme Click Nest to continue Hydroshear Plus User Manual Page 23 DIG L B Chapter 3 Installing the Hydroshear Plus Select the radio button Install the software automatically and click Next to continue Found New Hardware Wizard The wazad helps you install software for USB4o Senal Port Drier If your hardware came wilh an installation CD lt E floppy disk insert CO Install rom oc specific locaton Advanced Lick Hest bo continue Be Select the driver in the location C windows inf oem22 inf and click Next Found New Hardware Wizard Please select the best match for your hardware from the list below E LISB to Serial Port Driver i facturer Location ip Semiconductor Technology c windows inf oem22 inf Semiconductor Technology d
62. on 3 Dilute Tris EDTA buffer 100 x concentrate 100 fold with ultra pure water 100 x Tris EDTA buffer concentrate available from Sigma 19285 Ulta Pure water available from BioChemica 95284 or Sigma 95284 Never use tap water All final diluted solutions should be filter sterilized using either 0 45um or 0 2 um filter available from Millipore Pall or Sartorius It is recommended to purchase the Wash Solution Bottle Kit from Digilab part number HSPO0040 The Digilab Wash Solution Bottle Kit includes one each 250ml bottles The Digilab Wash Bottle Solution Kit comes with three 3 caps with holes for the 1 16 tubing to go through in order to prevent particulates and reduce any contamination and are color coded to match the colored labels on the corresponding tubing Also included is a piece of tubing which goes to waste one empty waste bottle All four bottles can be placed in the Hydroshear bottle holder which 1 part of the base of the Hydroshear Plus GIL Y DIGIL B IA y E ue Serong UEM 5992 22010 Deli OX Em Gloves Hydroshear Plus User Manual Page 27 4 25 AS UR aM Chapter 3 Installing the Hydroshear Plus 3 5 INSTALLATION Take care when working with the syringe although the Hydroshear Plus syringe 15 designed for higher pressure it is made of elass and could shatter under high pressure Wear protective eyewear whenever the plastic door to gain access to the syring
63. or written consent of Company Trademarks shall be used in accordance with accepted trademark practice including identification of trademark owners names These Term and Conditions do not grant the Customer any intellectual property rights in this Software DIGILAB Contents GENERAL MOLIS P E ERR m I CONTACTING DIGIISAB E vus asi I LEGAL INFORMATION issue Eo CHAPTER1 ABOUT THE HYDROSHEAR PLUS PRODUCT WARRANTY 7 ra COE ECT 8 TECINICAE SPECIFICATIONS 8 COMPUTER SOFTWARE REQUIREMENTS cccccccsssscccssscccccscsccccsccccccccccccccccccccecccccccescsccccsccccceeess 8 ELECTRICAL REQUPRE VEN US ciceccseccbetcenccsecessdeccsncceceeesdeccosssceusensaccsssscscsete lt ases lt seescoxssesecssdeocesesseee 9 ENVIRONMENTAL REQUIREMENTS ccccccsssscccsscsccccsccccccssccccccscsccccsccccccccccccccscscccsscsccscsccccsceccccss 9 CHAPTER2 HOW THE HYDROSHEAR PLUS WORKS 2 1 2 2 2 3 SHEARING ASSEMBLY ccscccncccuchvcexcaaseeccsecs scsdesswnnscdeenseaseussasenceessaadeecsssestiesbvecesescenscdeasencesnssssescdexsus 11 UV T N
64. ould point toward the Hydroshear Plus valve 9 Metal ferrule the tapered end of this ferrule should be close to the end of the orange tubing and should point toward the Hydroshear Plus valve Hydroshear Plus User Manual Page 32 DIGILAB Chapter 3 Installing the Hydroshear Plus 3 6 2 DETERMINING THE ORIENTATION OF AN UNLABELED ORIFICE BOX 1 Unscrew the input output nuts and their associated tubing from the orifice box 2 inside the ends of the orifice box to observe the ruby which appears as a pinkish circle 3 One side of the ruby will appear smaller and less reflective than the other this is the output side of the orifice box INPUT OUTPUT Figure 3 3 Determining correct shearing assembly orientation 4 Screw the output tubing nut into the output port and the input tubing nut into the input port 5 The shearing assembly 15 now ready for use Hydroshear Plus User Manual Page 33 DIG L B Chapter 3 Installing the Hydroshear Plus 3 6 3 ATTACHING THE SHEARING ASSEMBLY TO THE HYDROSHEAR PLUS 1 Check to see that the components of the shearing assembly are correctly oriented as described above Figure 3 2 Shearing assembly components 2 Attach the shearing assembly to the port by tightening the base screw into default output port or to the location you have chosen in the software Figure 3 4 Attaching shearing assembly to the Hydroshear Plus 3 Tighten base
65. over wash solution may damage or destroy DNA 8 2 2 SIZE OF SHEARED DNA FRAGMENTS IS INCORRECT There are three likely causes of incorrect fragment size 1 Air bubbles were in the sample during calibration or sample shearing During the next shearing run carefully follow the step by step instructions in section 5 3 Shearing DNA Samples This procedure includes the steps necessary to remove air bubbles from the sample If the problem persists after you have verified that bubbles are not the problem check possible causes 2 and 3 below Hydroshear Plus User Manual Page 74 DIG L B Chapter 8 Troubleshooting 2 The shearing orifice is partially clogged Unclog the shearing assembly See section 7 4 Unclogging Shearing Orifice Boxes using Sonication If you follow the unclogging procedure and the fragments produced are still the wrong size recalibrate the shearing assembly See section 3 6 4 Calibrating the Shearing Assembly If after recalibrating the shearing assembly the fragments produced do not correspond to the speed codes determined during the calibration procedure this is not the problem and you should proceed to possible cause 3 below 3 The shearing assembly may have been dropped or mishandled and damaged Discard the shearing assembly Calibrate and then use a new shearing assembly 8 2 3 THE FRAGMENTS PRODUCED DO NOT CLONE WELL Poor cloning can be caused by many factors unrelated to the s
66. r the buffer normally used to resuspend the DNA samples Starting with a speed code of 10 step down one in a series of subsequent runs These shearing runs should be performed using 30 cycles so if there 1 a slow leak it will be detected When performing these test runs click No when the Hydroshear Plus software asks Do you want to wash the device before starting the shearing process Continue until liquid can be observed leaking from the shearing assembly connections or until the system gives a Plunger Overload error Since this speed code is obviously too low shearing should only be attempted using a speed code that 1s at least one level higher For example if the system fails at speed code 6 then the limit for a shearing run would be speed code 7 NOTE The maximum speed code for a small shearing assembly 15 generally 6 and the maximum speed code for a standard shearing assembly 15 generally 3 4 3 6 STEP DOWN SHEARING METHOD If a sample 1s to be sheared at or near the speed code limit it may be necessary to initially shear the sample at a higher speed code i e with the syringe moving more slowly followed by re shearing the same sample at the low speed code This will vary depending on the samples being sheared For example shearing at a speed code of 16 for 20 cycles eject the sample then reload the sample and shear at a lower speed code of 4 6 for 20 cycles Note that if the speed code entered is less than 20 the software
67. roshear Plus User Manual Page 70 DIGIL Chapter 7 Maintenance 7 5 2 REPLACING THE HYDROSHEAR PLUS INPUT AND WASH TUBING 1 Using the 1 4 hex wrench provided in the Hydroshear Plus tool kit loosen the hex screw that attaches the input tubing to the valve Use your fingers to finish unscrewing the hex screw from the valve 2 Pull the old tubing out of the hex screw Figure 7 5 Sample input tubing 3 Insert the end of the plastic tubing without the metal pressure fitting attached into the threaded end of the hex screw The tapered end of the metal ferrule should face away from the threaded end of the hex screw as shown 4 Insert the ferrule of the new input tubing into the port on the right side of the valve 5 Screw the hex screw into the port tightening with the 1 4 hex wrench 7 5 3 REMOVING THE HYDROSHEAR PLUS SYRINGE 1 Using the Manual Operation window draw 500 ul of air into the syringe See Section 6 2 Manual Aspiration into the Hydroshear Plus Syringe The syringe plunger cannot be manually pulled down with the Hydroshear Plus turned off 2 Turn the Hydroshear Plus off using the power switch on the back of the instrument and disconnect the power cable from the wall outlet 3 Reverse the procedure as described in Section 3 5 Installation of the syringe Hydroshear Plus User Manual Page 71 DIGIL Chapter 7 Maintenance 7 6 DRIVE SCREW MAINTENANCE If the p
68. s 3 3 HARDWARE INSTALLATION 1 Plug the serial cable connectors into the ports on the back of the Hydroshear Plus It does not matter which connector goes to which port on the Hydroshear Plus as the software will guide you to select the appropriate COM ports and instruct to switch the serial cable if necessary See 4 1 Starting the Hydroshear Plus Software Power Cord Two Serial Connectors Figure 3 1 Rear view of Hydroshear Plus 2 Secure the serial cable to the Hydroshear Plus by tightening the screws on either side of the connectors 4 Plug the USB cable connector into a USB port on the CPU 5 Plug one end of the power cord into the back of Hydroshear Plus and the other end of the power cord into a properly grounded wall outlet 6 Turn the Hydroshear Plus power switch on the back of the instrument to the on position 1 ON 0 OFF Hydroshear Plus User Manual Page 26 DIG L B Chapter 3 Installing the Hydroshear Plus 3 4 WASH SOLUTIONS There are three Wash Solutions used with the Hydroshear Plus Wash Solution 1 0 2M HCL Wash Solution 2 Base 0 2M NaOH Wash Solution 3 pH 8 0 Wash Solution 1 x Concentration of TE Buffer Wash Solutions Formulations Wash Solution 1 Dilute IM HCL Hydrochloric Acid 5 fold with ultra pure water Wash Solution 2 Dilute 10 M NaOH Sodium Hydroxide 50 fold with ultra pure water 10 M NaOH available from BioUltra 72068 or Sigma 72068 Wash Soluti
69. s design 15 based on the hydrodynamic point sink shearing method originally developed by Oefner et al Nucleic Acids Research 24 3879 3886 1996 a theoretical model of hydrodynamic flow through such a system Strictly speaking it 1s the rate of strain tensor that describes the force on a molecule and hence its breakage In the present application the breakage is caused by both the shearing forces when the fluid 1 inside the narrow tube or orifice and the extensional strain force when the fluid approaches the orifice Thorstenson et al Genome Research 8 Issue 8 848 855 Aug 1998 Direction of flow Figure 2 1 How the Hydroshear Plus Works The action of the syringe pump plunger forces the DNA 1n solution towards the orifice Because the orifice 1 smaller than the tubing leading to it there 1 an abrupt contraction in the diameter of the fluid s path As the solution 1s forced through the small opening the pressure of the solution in the mechanism builds before the orifice and then drops dramatically after passing into the orifice This dramatic pressure drop in turn compels the solution to accelerate 1n order to maintain its volumetric flow rate 1n accordance with Bernoulli s Equation for Frictionless Flow The acceleration of the solution creates drag forces i e extensional strain forces that stretch the DNA until its molecular bonds begin to break and the DNA snaps into fragments Fragmenting of the DNA continues unt
70. ssccccsssccccssccccsssccccssscccssscccessscoees 85 FIGURE 9 5 SHEARING RESULTS ARE INDEPENDENT OF STARTING FRAGMENT LENGTH e eee ee eee eee eoe eoooo 86 FIGURE 9 6 SHEARING RESULTS INDEPENDENT OF STARTING FRAGMENT VOLUME cccsccsssssccccssccccssscoees 87 FIGURE 9 7 SHEARING RESULTS ARE INDEPENDENT OF STARTING FRAGMENT CONCENTRATION ccssscccssscoees 88 V1 DIG L B Chapter 1 About the Hydroshear Plus Chapter 1 ABOUT THE HYDROSHEAR PLUS The Hydroshear Plus is an automated Point Sink Shearer offering a simple reproducible and controllable method of fragmenting DNA The Hydroshear Plus creates and controls hydrodynamic forces that work in conjunction with its innovative shearing assembly to shear DNA A wide range of DNA samples are compatible with the Hydroshear Plus and the shearing parameters can be adjusted to produce specific fragment lengths of DNA 1 1 PRODUCT WARRANTY INFORMATION IMPORTANT The Hydroshear Plus 15 shipped with a Product Warranty Card that needs to be filled out and sent back to Digilab in order to be placed in the Digilab customer database and to initiate the Warranty The Hydroshear Plus Limited Warranty 1s as follows LIMITED WARRANTY Seller warrants that all Products consisting of new equipment shall be free of defects in material and workmanship under normal use and service for a period of one 1 year after the date of shipment Seller warrants that all consumable Produ
71. sure it is not too low for the appropriate shearing assembly which are 7 for the small shearing assembly and 3 for the large or standard shearing assemblies 8 3 11 OTHER ERROR MESSAGES Miscellaneous error messages will appear if the pump receives commands from the software that are illogical The majority of these errors can be resolved by reinitializing the pump as described in section 6 1 Explanation of Manual Operation Controls If reinitializing the pump 1 necessary when your sample 1 still within the syringe place a collection tube at the sample input tubing prior to performing this step Hydroshear Plus User Manual Page 81 DIG L B Chapter 9 Performance Data Chapter 9 PERFORMANCE DATA 9 1 RELATIONSHIP OF FRAGMENT SIZE TO PUMP SPEED The fragment size produced by the Hydroshear Plus 1 correlated with the pump speed The pump speed for a shearing run 1 designated by the user The higher the pump speed the slower the syringe pump moves Method Several samples of bacteriophage lambda DNA were sheared at different speed codes The sheared samples were run on an agarose gel to determine the resulting fragment sizes Result As speed code value was decreased i e as pump speed increased the fragment size produced decreased Thus fragment size 1 inversely correlated with pump speed and is directly correlated with the speed code 8 9 1011 E Speed code 500bp ladder 3 5 7 9 11 13 15
72. tes Pour the isopropyl alcohol out of the flask leaving the orifice boxes inside Add ultrapure water to the flask swirl gently and then discard the water Add fresh ultrapure water to the flask and inspect for air bubbles Place the flask into the sonicator and sonicate for 10 15 minutes Remove the orifice box discard the water and let the orifice box dry for at least 30 minutes Hydroshear Plus User Manual Page 69 DIG L B Chapter 7 Maintenance 7 5 REPLACING HYDROSHEAR PLUS TUBING In order to ensure proper shearing the Hydroshear Plus tubing must be kept clean and free from blockages and crimps Dirty blocked or crimped tubing can cause various problems including poor accuracy loss of precision and syringe stalling or break down To replace the tubing for your Hydroshear Plus contact Digilab to order new tubing 7 5 1 REPLACING THE HYDROSHEAR PLUS OUTPUT TUBING Each new shearing assembly has the output tubing pre attached If necessary you can replace the output tubing without replacing the entire shearing assembly Refer to Figure 7 4 Output Tubing and Orifice Box and follow the instructions below 1 Unscrew the black plastic output tubing nut from the orifice box 2 Push the new output tubing through the black plastic output tubing nut 3 Attach the output tubing nut to the orifice box by screwing it into the output port Do not over tighten Figure 7 4 Output Tubing and Orifice Box Hyd
73. th a standard shearing assembly unit at speed codes raising from 3 19 left to right Hydroshear Plus User Manual Page 35 DIG L B Chapter 3 Installing the Hydroshear Plus P23 45678 9100 4 5000 bp 3500 bp 2500 bp 2000 bp 1500 bp 1000 bp 15 agarose gel run at 145V for 2 hours All samples are 2 ug 100 ul lambda Figure 3 5 Agarose gel of DNA sheared at different speed codes 5 tables below show the typical size ranges achieved by the standard small and large shearing assemblies Table 1 Typical speed codes for standard assembly TY 3 D 12 15 Table 2 Typical speed codes for large and small assembly Hydroshear Plus User Manual Page 36 DIGILAB Chapter 4 Using the Hydroshear Plus Software Chapter 4 USING THE HYDROSHEAR PLUS SOFTWARE 4 1 STARTING THE HYDROSHEAR PLUS SOFTWARE 1 To start the Hydroshear Plus software locate the program titled Hydroshear Plus in the Windows Start gt Programs menu 2 The dialog box shown below appears informing you that shearing parameters should be changed if you do not want to use the default settings Machine parameters are currently set to the Factory defaults IF vau don t wish use the Factory settings vau should specify other values For the machine parameters before continuing 3 Click OK to dismiss the dialog box Shearing parameters are currently s
74. th the syringe fully lowered for 30 minutes Hydroshear Plus User Manual Page 64 DIGILAB Chapter 7 Maintenance 3 After the 30 minute period remove the input tubing from the detergent 4 the fluid from the syringe into a waste container 5 Leaving the inlet tubing open to air fill the Hydroshear Plus syringe and then eject into an appropriate waste container 6 _ and then empty the Hydroshear Plus syringe with distilled or deionized water for at least 10 cycles 7 Leave the input tube open to air And fill the Hydroshear Plus syringe and then eject into an appropriate waste container Repeat procedure 3 times Tele CLEANING WITH WEAK BASE AND ACID IN SEQUENCE 1 Fill the Hydroshear Plus syringe with Wash Solution 2 2 Allow Wash Solution 2 to remain in the syringe with the syringe fully lowered for 10 minutes 3 After the 10 minute period remove the reagent tubing from the Wash Solution 2 4 Fill and then empty the Hydroshear Plus syringe once with distilled or deionized water 5 Fill the Hydroshear Plus syringe with Wash Solution 1 6 Allow Wash Solution 2 to remain in the pump with the syringe fully lowered for 10 minutes 7 After the 10 minute period remove the inlet tubing from the Wash Solution 1 8 Leaving the inlet tubing open to air fill the Hydroshear Plus syringe and then eject into an appropriate waste container 9 Hil and then empty the Hydroshear Plus
75. to get a copy of the manual for the USB to 2X RS232 cable double click on manual and then select the Icon that says WIN 2000 XP EN Hydroshear Plus User Manual Page 16 DIGILAB USB TO 2 RS232 Cable File Edit view Favorites Tools Help Back 27 Search lg Address D USB TO 2 x R5232 Cable ECT Files Currently on the CD Folders 8 O drivers amp EXACT C3 f48ad320f524827469bd79b72F amp 186 Linux mac 73 C3 MyIDM Program Files 3 Qoobox E C3 SwDist O temp WINDOWS 3 Driver D USB2 0 EASY TRANSFER LINK CABLE FOF O USB 2 0 Easy Link Net Cable USB 2 0 Network Cable USB 2 0 TO R5232 Cable USB 2 0 TO R5232 Converter USB Link Cable E O USB Network Cable USB TO 2 x R5232 Cable D Linux mac manual 8 C3 windows USB TO 2 x R5422 Cable USB TO Parallel Printer Cable manual lt Chapter 3 Installing the Hydroshear Plus 2j windows In order to install the drivers for the Hydroshear Plus cable in the same screen as above select and double click on windows as shown below USB TO 2 RS232 Cable File Edit View Favorites Tools Help Back o lt 27 O Search e Folders Address D USB TO 2 x 5232 Cable Folders drivers EXACT F48ad320f524827469bd79b72f 1386 Linux mac E 73
76. ump is making a screeching noise or if the syringe 1 stalling frequently the drive screw needs to be lubricated Lubrication 1s usually only needed after 700 000 syringe strokes If more frequent lubrication is required there may be another problem To lubricate the lead screw use a cotton swab to spread a generous layer of gear lubricant along the lead screw P 2 PE Figure 7 6 Lubricating the Drive screw Use the Manual Operation controls see Chapter 7 Manual Operation to retract and expel fluid or air from the syringe several times The lubricant will be spread along the lead screw during operation of the syringe Hydroshear Plus User Manual Page 72 DIG L B Chapter 8 Troubleshooting Chapter 8 TROUBLESHOOTING 8 1 PROBLEMS OBSERVED DURING SHEARING RUNS 8 1 1 AIR BUBBLES IN THE SYRINGE This 1s normal When fluids are drawn into the Hydroshear Plus syringe air bubbles sometimes form The software will ask you 1f bubbles are present and guide you through the necessary steps to remove them as part of the shearing run procedure see Section 5 3 Shearing DNA Samples 8 1 2 CLOGGED SHEARING ASSEMBLY If any of the following symptoms are apparent during a shearing run then a blocked or partially blocked shearing assembly 15 the likely problem A Air bubbles in the syringe when retracting sample from the output tubing Frothy bubbles in the output tubing sa
77. ut prior notice to holders of the edition The reader should consult Digilab if errors are suspected IN NO EVENT SHALL DIGILAB BE LIABLE FOR ANY DAMAGE ARISING OUT OF OR RELATED TO THIS DOCUMENT OR THE INFORMATION CONTAINED IN IT Trademarks Digilab Hydroshear Plus DNA Shearing Device Digilab are registered trademarks of Genomic Instrumentation Services Inc in the U S and some countries Hydroshear Plus DNA Shearing Device is a trademark of Genomic Instrumentation Services Inc Windows is a registered trademark of Microsoft Corporation Pentium and Pentium registered trademarks of Intel Corporation Digilab Software License General All sales by Genomic Instrumentation Services Inc the Company of its products including hardware components Instrument and software Software incorporated therein collectively the Products to its original owner Customers are subject to the following terms and conditions Terms and Conditions Instrument means the tangible physical components of Products specifically the Hydroshear Plus DNA Shearing Device Software means any software or related documentation incorporated into the Products or provided under a separate agreement Customer means any person who purchases Products directly from Company or one of its authorized distributors for internal use and is the original owner of the Product Software license The software is
78. want a slow syringe movement y Click the Start button 8 One pump pass is conducted at the specified speed code and the specified volume is ejected from the Hydroshear Plus syringe Hydroshear Plus User Manual Page 63 DIGIL Chapter 7 Maintenance Chapter 7 MAINTENANCE 7 1 GENERAL MAINTENANCE GUIDELINES Keep Hydroshear Plus covered when not in use dust can reduce the life of the valve Wash the Hydroshear Plus after each use The Hydroshear Plus syringe pump should not be run dry for more than a few cycles Observe the Hydroshear Plus syringe pump for leaks correct any problems immediately Wipe up spills on and around the Hydroshear Plus immediately especially wash solutions which could contain acidic or basic solutions 7 2 PERIODIC CLEANING The cleaning procedures listed below are included as options for users who feel that their machine requires extensive cleaning or have neglected the daily maintenance tasks However if you perform the general maintenance steps outlined above the more rigorous cleaning methods should not be required For instructions on drawing fluid or ejecting fluid from the Hydroshear Plus see Chapter 6 Manual Operations 7 2 1 CLEANING WITH WEAK DETERGENT l Use the Manual Operation controls to fill the Hydroshear Plus syringe with weak detergent solution Leave the input tubing immersed in the detergent 2 Allow the detergent to remain in the pump wi
79. yringe 5 movement Please also refer to section 4 3 5 Finding the Maximum Speed Code for a description of the procedure for determining the maximum speed code i e the maximum syringe speed that an individual shearing assembly can withstand Hydroshear Plus User Manual Page 77 DIG L B Chapter 8 Troubleshooting 8 3 6 RESIDUAL VOLUME In current versions of the Hydroshear Plus software the residual volume is the volume that is not recovered after the sample is ejected from the syringe This value is approx 16 20 ul If you are concerned about sample loss note that it is possible to dilute your sample into a larger volume prior to shearing This will reduce the amount of DNA lost during the shearing process Following the shearing run the DNA sample can be precipitated and concentrated for downstream applications 8 3 7 PORT NUMBER The Port Number corresponds to the CPU serial port to which the Hydroshear Plus 1 connected The possible CPU ports used and the corresponding Port Number are listed underneath the Port Number heading and value text box e g 0 COMI If an incorrect Port Number is entered an error 37 message will appear when the Hydroshear Plus software starts up Error 37 occurred at Serial Pork Init Possible reasons LabVIEW Device nat Found Clicking OK on this error message window may bring up additional error messages Serial error while Read timeout while att

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