User Manual - Sage Science
Contents
1. Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com Increment Increment 2000 1500 msec Step 1000 Step Time Forward 500 x Step Time Reverse 0 Fig 2 An example of second increments added 5 msec was added to the forward increment and 3 msec increment was added to the reverse increment 4 cycles 2000 1500 msec Step 1000 Step Time Forward 500 x Step Time Reverse 0 Fig 3 An Illustration of running four cycles of step of the previous protocol In this example each cycle is about 6 seconds in length Typical runs require 8 16 hours Note If the reverse time is not long enough large molecules may not completely change direction during the reversal and may actually migrate faster than shorter molecules This effect can be completely overcome by using a time ramp whose longest reverse time is long enough to separate all molecules of interest sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 3 0 Using the Pippin Pulse The Pippin Pulse software includes three pre set protocols based on internal testing These are a good starting point to analyze fragments from 0 5 80 kb Go to the Pre set Protocols section of this manual Section 6 for the buffer formulation and conditions for these runs Goto Programming the Pippin Pulse Section 9 for recommendations and guidelines for developing2. Pulse User Manual Rev F 978 922 1832 WWW Sagescience com 6 3 Pre set protocol example 2 1 50kb Run time 9 hours Voltage 80 V Buffer 0 5X KBB see page 7 Agarose 0 7596 Lonza SeaKem GOLD 110 ml z a 8 17 0 em 15 0 12 2 g5 an 8 Gem z e 6 o A o d 3 2 e 1ko WE sage science Pippin Pulse User Manual Rev F support sagescience com 10 978 922 1832 www sagescience com 6 4 Pre set protocol example 3 3 70kb Run time 14 hours Voltage 75V Buffer 0 5X TBE see page 8 Agarose 1 Lonza SeaKem GOLD 110 ml Q sage science Pippin Pulse User Manual Rev F support sagescience com 11 978 922 1832 WWW Sagescience com support sagescience com 7 0 Running the Pippin Pulse 1 Setupthe complete gel system load the DNA samples and connect the electrodes to the Pippin Pulse 2 Turn the Pippin Pulse on by pressing the power button on the front panel 3 Launch the Pippin Pulse application by double clicking the Pippin Pulse Icon 4 Using your mouse pointer select the protocol or Waveform Type name to be run The selected protocol will be highlighted in blue Target Duration hh mm Bn US OU 6 Press START sage science Pippin Pulse User Manual Rev F 12 978 922 1832 WWW Sagescience com support sagescience com 8 0 Monitoring a Pippin Pulse Run 1 When running the Pippin Pulse High Voltage
3. Status will indicate a yellow color 2 Therun voltage current values and run progress will be displayed High Voltage Status Progress hh mm ss 01 11 43 8 1 Ending a Run To abort a run before the target duration press the ABORT button 8 2 Log Files A log file is saved after every run The file name is saved as PP year month day hour min sec txt The files are saved in a sub folder titled Pippin Pulse that is automatically placed in the computer s Documents folder during software installation The file lists the measured voltage and current values at 1 second intervals for the entire run e Public Documents SageScience Poppin Pulse Logs 2012 12 oD enm DI Organiza include im library Share wth Bum New folder 1 D Favorftes xia WE Desktop e PE 2017 12 18 17 22 54 8 Download e HP 2012 17 19 15 52 16 T s v v I mA 1 24 2 1 18 2 24 3 1 54 3 24 7 1 54 4 24 8 1 85 13 sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 9 0 Programming the Pippin Pulse 9 1 What is a Pippin Pulse protocol A Pippin Pulse protocol is a series of parameters that control the run voltage forward and reverse time steps and number of steps per cycle A more complete explanation of these parameters is described earlier in this manual About Pulsed Field Electrophoresis Section 3 The functions of the p
4. and forth in the gel effectively slowing down large DNA fragments that might otherwise run at the same rate as smaller ones By switching the direction of the electric field in a gel DNA will change its direction of migration Since smaller molecules can change direction faster than larger molecules more differentiated separation can be achieved by rapidly switching or pulsing the electric field A single pulse duration typically separates a relatively narrow range of DNA sizes For best results it is necessary to use a range of pulse times this is accomplished by using a ramp progressively increasing the forward and or reverse intervals from a lower limit to an upper limit The Pippin Pulse is programmed by entering a forward time step and reverse time step A ramp is programmed by adding a time increment to either or both steps An accelerated ramp can be achieved by adding additional time increment either or both increments Lastly the number of time steps is also entered and the steps will continuously cycle for the duration of the run Cycling allows the same protocol to be run for different lengths of time reproducibly Increment 600 500 400 msec Step 300 Step Time Forward 200 100 X X Step Time Reverse 0 Fig 1 An example of time increments for the forward and reverse steps A 25 msec forward step with a 25 msec increment 10 msec reverse step with a 10 msec increment 20 Steps cycle Q sage science
5. User Manual Pippin Pulse Electrophoresis Power Supply Software Version 1 31 Part No PPIO200 978 922 1832 WWW Sagescience com support sagescience com Sage Science Inc Suite 3150 500 Cummings Center Beverly MA 01915 2013 Sage Science Inc All rights reserved Sage Science Pippin Prep BluePippin and Pippin Pulse are trademarks of Sage Science Inc SeaKem is a trademark of Lonza Group Ltd All other brands and name mentioned herein are property of their owners Q sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com Table of Contents COMTO o PT TOTO mmm 3 UM Ter eec PTT Ts 3 DZ WAG ETC T 3 2 0 About Pu lsed Field egene 4 3 0 Using the Oslo MET 6 MO recie AS Ent 6 SX e MM H 6 DEE 7 6 1 Recommended gel conditions ssasviviiivid evoriaia daos Tib EEl viae raisso s riens r siini EES E EI ESAE NEI TEISS 7 6 1 1 KBB Gel pH 8 7 5 15kb and LOOK 7 6 1 2 IR Ce AR e Ai e WEE 8 6 2 Pre set protocol example 59 5KD E RD SVO GE Fui SeE ERREUR RU Poe REIR IRI EE ESTA NI SV EUREN rade 9 6 3 Pre set protocol example e KT WEE 10 bob Preser protocol Enter de E e WEE 11 FO IAI the Pipon Ee 12 8 0 Monitore a Pippin Pulse RUD EE 13 Gute Tt S E 13 XM Loe STET 13 9 0 Programming the Pippin E 14 9 1 What is a Pippin Pulse protocol ssssesssssesesesesersrsesesrs
6. arameters are listed below and appear on the Editing screen of the Pippin Pulse software application V Voltage Electrophoresis voltage accept values between 25 150 V A Forward Time at start of run 1 65 535 msec B Reverse Time at start of run 1 65 535 msec C Increment added to A at each step 0 255 msec D Increment added to B at each step 0 255 msec E Increment added to C at each step 0 255 msec F Increment added to D at each step O 255 msec G Number of steps per cycle 1 65 535 9 2 Where to start The Table below may be used on a guideline to begin developing protocols for gel analysis of large DNA These guidelines are based on 5 10 V cm gels and 0 5X electrophoresis buffer see Pre set Protocols Section 6 Running the Pippin Pulse The protocols that are highlighted in gray are pre set in the Pippin Pulse software the remaining protocols have not been tested but are provided as a starting Approx Resolved Size Range kb Run Time recommendation Pippin Pulse Parameters A B C D E F G 2 50 10 50 10 0 0 20 1 30 8 hr 3 25 10 25 10 10 4 9 1 30 16 hr 6 150 50 30 10 3 1 81 10 200 20 hr 7 300 100 30 10 30 10 45 20 400 20 hr 8 300 100 300 100 0 0 100 20 800 20 hr 9 300 100 0 0 60 20 51 40 1000 20 hr 10 300 100 300 100 0 0 250 40 2000 20 hr Fig 4 Pre set protocols highlighted in gray have been validated by Sage Science Other protocols are suggested star
7. essrsesesesesererseseseserersesrseserersesesesereeeesesese 14 B2 WHOEEHO E GE 14 9 3 Programming the Pippin PUISE EE 15 10 0 Software Overview eese enne nennen REAN SAE aN ANE E EENE EEEE ARENE a Eanna 17 ffe TVS MIIN OEE EE 17 i0 MAE Ma E lte EE 18 11 0 PIBBIIEPUISS BIOD E 19 Q sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 1 0 Introduction Thank you for purchasing the Pippin Pulse electrophoresis power supply The Pippin Pulse provides programmable pulsed field power for running small to mid sized agarose gels A PC running Pippin Pulse software is required The PC and gel tank must be provided by the user 1 1 Specifications Electrophoresis voltage 25 175 V Maximum current 200 mA Dimensions 7H X 20W X 24D cm 3H X 8W X 10D in Weight 1 1 kg 2 5 Ibs 1 2 Warranty The Pippin Pulse is subject to depot repair or replacement at the discretion of Sage Science The warranty period is 1 year from the date of shipment Q sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 2 0 About Pulsed Field Electrophoresis Pulsed field gel electrophoresis is a strategy for resolving large fragments of DNA for analysis When running a typical direct current agarose gel fragments above 10 15kb can migrate in a non predictable manner that is not related to their size Pulsed field gels work by shuttling DNA back
8. he Main Screen lists the electrophoresis protocols provides run controls and displays the run status e The Edit Screen allows the programming editing and deleting of electrophoresis protocols 10 1 The Main Screen When the Pippin Pulse application has been started the following screen will launch Electrophoresis Protocol List Waveform Table barge UU auorT HP High Voltage Gaas Measured Amplitude V Maasured Current mA Progress hh mm ss EE eee EHE eee sar Ze ts Ww Run Time Run Controls Run Status set by user 17 sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 10 2 The Edit Screen The Edit Screen is accessed by pressing the EDIT button and will appear over the Main Screen Electrophoresis Protocol List J Edit User Waveforms phere so OU volt Increment added to 5 at eat ee Spee Forfvard time at start of run 1 j5 mse Increment added to C ate 255 msec Darse time at start of run 1 Increment added to Da MJ wq Ki SoS KAS RACH PO LYLE W Ze ep E C Inclement added EGRE Ta ste ES CSR Editing field Description of Editing commands waveform parameters for programming sage science Pippin Pulse User Manual Rev F 18 978 922 1832 WWW Sagescience com support sagescience com 11 0 Pippin Pulse Blog A blog on Pippin Pulse can be found at the sage science website at http www sagesc
9. ience com blog tips pippin pulse A link to this blog can also be found on the Pippin Pulse product page or by entering Pippin Pulse into the site s search bar We will endeavor post the following e new protocols that we ve developed e references on gel electrophoresis and pulsed field e additional tools e user input and suggestions We invite you to check back periodically to see what s new sage science Pippin Pulse User Manual Rev F 19
10. new protocols 4 0 Unpacking The Pippin Pulse PPIO200 will arrive with the following components e Pippin Pulse electrophoresis power supply e Power supply e USB cable to connect PC to a Pippin Pulse e USB storage device contains the Pippin Pulse programming software e User manual this document 5 0 Set up e Remove the Pippin Pulse from its packaging e Connect the power supply to the unit and plug the power cable into an electrical outlet e Insert the USB cable into the USB port found in the back of the unit e Insert the other end of the USB cable into a USB port of a PC e Install the Pippin Pulse software on the PC Insert the USB storage device into an available port on a PC On the computer go to the USB device Computer Removable Disk E Open the Pippin Pulse folder Double click the setup executable file Follow the installer instructions After installation is complete re boot the computer O O 0 O O E The following icon will appear on the computer desktop Pippin Pulse Pippin Pulse Desktop Icon Double click the icon to launch the application Q sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 6 0 Pre set protocols There are three pre set pulsed field protocols listed in the Pippin Pulse software These are highlighted in gray in the Pippin Pulse software and cannot be modified or deleted and indicated a fragment range o
11. ting protocols for running gels between 5 10 V cm sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 9 3 Programming the Pippin Pulse 1 Turn the Pippin Pulse on by pressing the power button on the back panel 2 Launch the Pippin Pulse application by double clicking the Pippin Pulse Icon 3 Pressthe EDIT Button The editing screen will appear over the Main Screen 15 sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 4 Place the mouse cursor in the Editing Field Enter a name for the protocol under Waveform Type and add the values for each parameter New Protocol j MENOS MENU en SS Wavetorm Parameters Incre Increm Y m rie Gi WM 5 Ifa previously written protocol is on the protocol list clicking on the protocol will make it appear in the editing field and it may be modified 10 48 kb Version A 6 Press ADD REPLACE to add the new protocol to the list If the same name appears in the Waveform Type field the protocol will be replaced with the newer one Waveform Type 0 5 15 kb 7 Press ACCEPT to save the new list and return to the Main screen 16 sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 10 0 Software Overview There are two screens in the Pippin Pulse software e T
12. ver which DNA should be well resolved These protocols were developed by Sage Science specifically to work with higher size range workflows with the BluePippin DNA size selection system Pippin Pulse v 1 31 Waveform Table 1 50kb 3 70kb DC Mode DC Mode 25 6 1 Recommended gel conditions The pre set protocol gel images shown on the following pages were run using the following gel formulations in a 12 X 14 cm gel tank 6 1 1 KBB Gel pH 8 7 5 15kb and 1 50kb Note 10X KBB buffer can be purchased from Sage Science Part No KBB1001 0 5X KBB running 10X buffer buffer g liter mM Tris base TAPS free acid EDTA free acid Lonza Seakem GOLD dH20 10X KBB buffer Total 3 sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 6 1 2 TBE Gel pH 8 0 3 70kb 0 5X TBE running buffer 10X buffer Tris base Boric Acid 0 5M EDTA Total 0 5X TBE Buffer Lonza SeaKem GOLD dH20 10X TBE buffer Total 100 ml 196 Agarose sage science Pippin Pulse User Manual Rev F 978 922 1832 WWW Sagescience com support sagescience com 6 2 Pre set protocol example 1 5 15kb Run time 8 hours Voltage 100 V Buffer 0 5X KBB see page 7 Agarose 0 7596 Lonza SeaKem GOLD 110 ml 15k x am dani 10k 8k 6k DEN k 4k emm 3k EN em 2k ee c e 1k mmm kd sage science Pippin
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