Indy Users Guide
Contents
1. Th Thermocycler YD Ip _ OQO u Q U mD diy Idaho Technology Inc NDY Safety and Regulatory Information This symbol warns the user to operate the machine according to the instructions provided in this manual If used otherwise a potentially hazardous situation could result This symbol highlights tips on how to operate Indy more efficiently Afczr turning pwer aff This label placed above the fuse and power switch wat 3 sacomds befara resturting the insument alerts the user to wait three seconds before restarting TWwoeawwltaaem the instrument lt also contains the fuse requirements aA 254V ZAG for this machine Note Always maintain Indy in good working order If the instrument experiences an extreme environmental event return it to Idaho Technology for a complete service inspection See Appendix A for return instructions If Indy is used in a manner other than described in this manual it may impair equipment protection and performance Copyright 2001 Idaho Technology Inc Indy Air Thermocycler User s Manual June 2001 rev 3 All rights reserved Printed in the United States of America The information contained in this document is subject to change without notice Idaho Technology makes no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a part2. When comparing an Indy Air Thermocycler to Heat Block Thermocyclers the cycle times are two to three times longer using a Heat Block Thermocycler One major factor is that Indy uses air instead of a metal heat block to rapidly heat and cool its samples Because of air s low thermal mass it can be temperature cycled much more quickly than a metal block yielding shorter cycle times This allows Indy to spend less time ramping to its target temperatures and provide more accurate temperature uniformity among samples In addition to faster cycle times air thermocycling reduces the amount of non specific product made Indy Air Thermocycler Inside this Chapter Guidelines for Sample Loading 11 Setting Up Indy Chapter 2 CHAPTER Setting Up Indy POPC eee eee eee eee eee cee cece eee ee Choose a location for Indy that allows free air to flow around the machine and into the air intake underneath Avoid placing Indy on any material e g lab bench paper which may get caught in the intake or otherwise obstruct free air flow Plug the power cord into the Indy power supply located on the right side of the instrument Connect the power cord to a grounded surge suppressor to protect Indy from electrical power fluctuations Avoid turning the Indy unit off and on abruptly When turning off the machine wait three to five seconds before turning it back on to ensure dissipation of any electrical charge still circul
3. Way Warranty Salt Lake City UT 84108 Non Warranty USA Date RMA Return Materials Authorization Company Department PO Phone Fax Instrument Serial No Notes Declaration of Decontamination Please read and completely fill out the Return Equipment and Decontamination Forms found at www idahotech com prod uct_sup forms htm The decontamination form and labels must be completed before the machine is returned to Idaho Technology In the event the unit is returned without documentation it will be shipped back to you at your expense Non warranty charges are currently 70 hr with a 2 hr minimum charge plus parts and shipping charges In the event the unit cannot be repaired it will either be returned to you or disposed of upon your request Thank you for your business and support of the health and well being of our employees Signature Date User must contact Idaho Technology prior to returning the instrument to receive an RMA 38 RETURN MATERIALS AUTHORIZATION FAX PG 2 OF 2 If you return an item to Idaho Technology you will be responsible for all return shipping charges Please note that your item must be returned with an Idaho Technology assigned RMA and Decontamination documentation Prior to returning the item you must contact Idaho Technology for an RMA number and also fax copies of all necessary Decontamination Forms to Fax 801 588 0507 Attn Technical Support All returns should be sent to the following
4. idahotech com Decontamination Forms amp Labels The following forms have been provided e Equipment Return Procedure e RMA Return Fax e Return amp Repair Form e Decontamination Labels Additional forms can be found on our 63 Web site www idahotech com For assistance please contact us by phone 801 735 6544 or 1 800 735 6544 36 EQUIPMENT RETURN PROCEDURE DECONTAMINATION INSTRUCTIONS TO BE FOLLOWED WHEN RETURNING EQUIPMENT TO IDAHO TECHNOLOGY FOR REPAIR OR REPLACEMENT Returned Equipment Equipment returned to Idaho Technology for repair and other reasons must be handled in a manner which will ensure minimal risk to the personnel involved in packing the unit for return and those responsible for receipt unpacking and processing at Idaho Technology Inc Note To ensure there is no loss of data any computers being retuned must be backed up prior to ship ment Idaho Technology is not responsible for any lost data ALL UNITS MUST BE DECONTAMINATED PRIOR TO BEING RETURNED TO IDAHO TECHNOLOGYINC The person responsible for the return must thoroughly survey the instrument for radioactive contamination and ensure its compliance to regulations A licensed person must complete the necessary forms and follow standard procedures by law Chemical Lab If a chemical lab makes the return the customer must thoroughly clean chemical contamination from the instru ment Clinical Lab If the instrument was used in a clini
5. is finished an End screen will appear showing you the program name and the time it took to run the pro gram ine Copy Lommand IS USed to Vupiicate a rrogram gt ia 1 Use the directional keypad and move the cursor to Copy 2 Press Enter and the Select user window will appear 3 Select the users name or adda new user Note If you plan to make changes to the duplicate program be sure to copy your program first then make the changes Aaaing Tet Pee eee eee eee eee ee eee ee ee ee eee ee eee eee eee ee ee 1 Press Enter to input the user name Indy Air Thermocycler 27 Programming Indy Chapter 4 2 Input the user name using the directional keypad and enter but ton Up to 9 Alphabetic letters and numerals can be used in a name 3 Select End and press Enter 4 The Select Program window will appear Press Enter to name the program 5 Inout the program name Select End and press Enter 6 To create a new program refer to Description of Programming Screen on page 22 in this chapter Note Select Esc to go to the previous screen at any time Chapter 4 Programming Indy 28 Indy Air Thermocycler Select User nn This function allows users to access any stored programs When the user selects this function a new display will list New User and any current user names Use the directional keypad to select a user name or new user Adding a New User cnn To add a n
6. thermocycling reaction Quick Run is designed to offer the user flexibility in programming runs in a simple and convenient format The Quick Run screen brackets the three segments e Summary 1 To enter this function use the directional pad to select Quick Run then select Enter 2 Edit the cycling parameters and program steps using the directional keypad 3 Load samples secure lid and select Run to begin Note Quick Run does not automatically save program data To save program data use the Copy function page 26 to create a new program 23 Description of Programming Screen In the Programming screen a brief hold step 94 C at 2 minutes precedes the default Run program denaturation annealing and extension Set Temperature Hold Time in Name of Cycle for Cycles min sec Program Number of cycles Slope Temperature Hold Step Line Denaturation Line Annealing Line Extension Line transition rate Program Controls The user can change the following cycling parameters C Number of cycles C Temperature in degrees Celsius Time Reaction time in minutes and seconds Slope Air temperature transition rate in degrees Celsius per second To edit any of these cycling parameters Use the directional keypad and move the cursor to the appropriate parameter and then use the numerical pad to enter the desired values To add a program segment Use the directional keypad and move the cursor
7. to the symbol of the desired segment Select Enter and a default segment will appear Input the new cycling parameters if necessary Chapter 4 Programming Indy 24 Indy Air Thermocycler To delete a program segment Use the directional keypad and move the cursor to the symbol of the desired segment Select Enter and the selected segment will delete Additionally the user can add or edit the following program steps to the first and last program segments Move the cursor to the of the first or last steps FirstSegment a LastSegment Select Enter Choose Seg Hold or Cycle gt Indy Air Thermocycler 25 Programming Indy Chapter 4 Choose Seg Hold or Cycle Select Enter Segment This adds an additional step in the existing cycle involving temperature time and rate Hold This adds a temperature hold at the beginning or end of the entire run Cycle A series of three steps denaturation annealing and extension Input desired temperature time and cycle numbers The Run Command is Used to Start a Program 1 Confirm the cycling parameters 2 Load the samples clockwise from position one and adjacent to each other and secure the lid 3 Use the directional keypad and move the cursor to Run 4 Confirm the sample volume to be run 25 ul or 50 ul select Enter to begin the reaction Chapter 4 Programming Indy 26 Indy Air Thermocycler When the Run
8. 0uL Display 128 x 64 pixel graphic display 30 Cycle Run Time 35 min with 25 uL sample vol 8 line 22 character 45 min with 50 uL sample vol backlit display Dimensions 20 cm x 15 cm x 23 cm Warranty Year parts and labor 30 day money back guarantee Blazing Speed Air cycling provides unmatched ramping and cooling rates making Indy two to three times faster than stan dard heat block thermocyclers These quick cycle times are achieved using standard 0 2 ml PCR sample tubes Compact Siz e The 20 cm x I5 cm x 23 cm footprint is small enough for every researcher to have an Indy in their per sonal work space Uniform and Accurate Temperature Control The instrument s cylindrical chamber provides sample to sample uniformity within 0 5 C The cham ber is heated via a resistive heating element and cooled with ambient air Quick Reaction Optinization The unparalleled speed of Indy Air Thermocycler allows quick optimization of reaction conditions and yields more specific product Simple Programming The 128 X 64 backlit screen and keypad allow quick and easy programming Even the most complex pro tocols are simple to program as operators are guided through a series of user friendly screens Oil F ree Operation The chamber is designed to prevent sample condensation thus eliminating the need for the addition of oil Idaho Technology Inc 390 Wakara Way Salt Lake City Utah 84108 USA 1 800 735 6544 ph 801 7
9. 36 6354 fax 801 588 0507 it idahotech com www idahotech com
10. Canada 1 801 736 6354 Utah IDD Prefix 1 801 736 6354 International Reach Us By Fax To contact Idaho Technology by fax use the following numbers 1 801 588 0507 United States and Canada IDD Prefix 1 801 588 0507 nternational Contents INTRODUCTION sca 25sistosnesacaansiceseatanilinpasuteninndeodaapsdasadsubobaiiapersteaeiaehginanteaensiadeleaeine Gata anieasioias 4 CHAPTER 1 POUT NAGY sxsientesiciieniasisbetccscce etna si ailed E S tecsauenaualeusevaiercesuenere 5 CHAPTER 2 Setn UD INS MNAY assacerakiero Rie coseiceas taney a ceca maaan doe tae eens 11 Storing and Protecting your Instrument Proper Sample Loading CHAPTER 3 Optimization Or nay REACTIONS sis c22jersiene ese User A E E 15 Converting from the RapidCycler and Heat Block Cyclers Optimizing a new set of Primers CHAPTER 4 Programming darnenn N a a EN 21 Quick Run Copy Programs Adding New Users Adding New Programs CHAPTER 5 Troubleshooting amp Maintenance ccccccccsssecccceeeeceeeeeeeeecceeuseeesseueceessegeeesssaeeeessaeeeeeeas 29 APPENDIX A Return and Decontamination FOrMS ccccccccseccceececeeeeceeeceeeeesueeeseeceseueeseeeseneeseeesseeess 35 APPENDIX B Waranty Sc Upgrades reon a N 42 INDEX zacit a ET E A E T 43 Introduction Idaho Technology is pleased to present the Indy Air Thermocycler our newest and most advanced air thermocycler The core of our system is ambient circulating air to rapidly heat and cool the samp
11. address Idaho Technology Inc 390 Wakara Way Salt Lake City UT 84108 USA hereby agree and accept all terms listed above Name Title Signature Date 39 REPAIR amp RETURN FORM THE FOLLOWING INFORMATION MUST BE COMPLETE TO ENSURE PROPER PROCESSING Equipment Information Return Materials Authorization number RMA Model Number Serial Number Use of equipment Description of Problem Customer Information Contact Name Company PO Exact return shipping address Decontamination Information This product must be decontaminated in accordance with an established government regular tion or equivalent rendering it free from radioactive biological or chemical contamination Which method was used What chemical infectious toxic or radioactive substances have been in contact with this product Also indicate if flammable or corrosive Authorization Notice By accepting this authorization to return this product the user assumes all responsibility for decontamination and cleaning __ Idaho Technology Inc reserves the right to refuse the delivery of products which do not appear to have been properly decontaminated If the equipment was used with or around radioactive material the signature of the safety officer is also required Date Signature 40 Pa 1 oF 2 DECONTAMINATION LABELS Please complete these one to the product and the other to the exterior of the shipping carton Fai
12. are model to calculate the sample temperature Air tem perature is measured using a fixed thermocouple inside the chamber and the software calculates the sample temperature Indy does not measure the temperature in the actual sample In order to heat and cool samples Indy utilizes an overshoot undershoot scheme where the air temperature briefly exceeds the target temperature This mathematical model determines how much and how long to over shoot undershoot the air temperatures This method ensures that samples achieve precise target temperatures When setting up a run users are asked the sample volume to help the soft ware calculate the proper target temperatures Since a 25ul sample heats and cools faster than a 50ul sample the air temperature does not over shoot undershoot as much or as long to heat cool the smaller volume sam ple For example to reach a denaturation temperature of 94 C the air temperature is quickly raised to approximately 115 C where it holds until the sample temperature nearly reaches the target temperature Just as the sample temperature reaches the target temperature the air temperature 10 drops to the target temperature where it is held for the duration of the pro gram Overshooting and undershooting target temperatures heat and cool the sample even faster than heating cooling the samples at target tempera ture only Comparison of Temperature Profiles Heat Block Thermocyclers Indy Air Thermocycler
13. ating in the system 12 keep this exhaust area clear Be sure to keep heat sensitive material and other volatile organic solvents way Heated air is expelled along the front and side of the lid it is important to The Indy sample chamber houses the samples in a circular array to ensure uniform temperature conditions The three sample blanks in the sample chamber maintain these conditions so if only a few tubes are run the first tube does not heat more quickly than the sec ond and third tube It is therefore important to load the PCR tubes clockwise starting in the position next to the sample blanks The control thermocouple which accurately measures the temper ature in the chamber is located in the chamber next to these sample blanks Load samples clockwise beginning at position 1 Guidelines for Sample Loading e Match the volume of the samples to the volume selected during pro gramming e We recommend a temperature hold at 94 C for one to two minutes before the first cycle of the denaturation temperature If the sample temperature differs from ambient temperatures by a large amount example the samples have been stored at low temperatures just prior to a run i e fridge or freezer Indy will not Know what the start ing temperature is Placing a temperature hold before the first cycle will allow the starting sample temperatures to equalize Indy Air Thermocycler 13 Setting Up Indy Chapter 2 e To
14. cal lab the person responsible for the return must thoroughly decontaminate the unit wiping it down with 0 5 NaOCl chlorine bleach diluted 1 part bleach to 9 parts water Use care not to mix with other chemicals mixture with acid will liberate chlorine gas Since the solution is somewhat caustic gloves and a face shield are recommended when preparing and using the solution When corrosion by NaOCl may be a problem a flush with water is permissible after the NaOCl has remained in contact for a minimum of 10 minutes Decontamination Labels After the above steps have been completed the person responsible for the return must complete and sign two Decontamination Labels and attach one to the instrument and the other to the exterior of the shipping container Complete and sign the Decontamination Form a photocopy should be made for your records and the original must be returned with the instrument Additional forms and labels can be found at our Web at www idahotech com NOTE A Return Materials Authorization Number RMA must be obtained from Idaho Technology prior to shipment and the RMA and Decontamination Labels must be visible on the exterior of the shipping container i Idaho Technology reserves the right to return and or refuse receipt of any materials at the customers expense that do not meet these requirements 37 RETURN MATERIALS AUTHORIZATION FAX PG 1 OF 2 From Pages Attn Ship to Idaho Technology Inc 390 Wakara
15. dular nature of Indy in the future it will be possible to upgrade the performance of both the hardware and software Any such upgrades will be offered at a reasonable cost with minimal time disruption Index Bovine Serum Albumin BSA 15 Condensation 0cccceee 15 Construction and Design 5 Copy Command 06 26 Customer Support 0 2 GY CHAO eA 25 Decontamination 0 35 Delete Program or User 28 Fuse Replacement 30 Heat Block Thermocyclers PE E E EE AA ian 10 18 Hold Temperature 12 25 KOVAO scuso 6 22 Load samples c 000008 13 Maintenance c cccceeeee 29 Master MiX ccccceeceeeeee es 17 New User cccccccecececececeess 26 43 Oil Overlay ceeeeeeeeeeeeeees 15 Optimization 0 15 19 Programming s0eceseeeeeeees 21 Quick Run ccs sesssswuteinicnsdsiacaans 22 RapidCycling protocols to Indy Saleisha ane tiraee 16 Return Procedure 36 Run Command 060008 25 Sample Loading 06 12 SEGMENT siendien 25 SOWING UD iaa 11 Technical Support 2 Temperature Profiles 10 Thermocouple 0000 30 Troubleshooting 29 33 Upgrades cccccseceeeeeeeeeees 42 WANA sompen a 42 Specifications Capacity 24 x 0 2 ml PCR tubes Fuse Requirements 8A 250V 3AG Sample Volume 25yL or 5
16. ensure uniform temper atures among samples load samples tubes clock wise starting at position 1 Load them directly adjacent to the previous sample as shown in the neighboring image and indicated on the top of the sample plate e Be sure the sample lids sit below the top lip of the Loading first sample in position 1 and do not space samples when performing a sample plate Note Do not put samples in any other slots other than the round 24 holes provided Doing so would block the fan and damage the instrument e Keep the lid closed when run ning Indy If the lid is opened during a run the heat coil will immediately shut off but the fan will continue to run Thermocycling will not contin ue until the lid is securely latched Note Opening the lid during a run may cause your reactions to fail 14 Indy Air Thermocycler 15 Optimization of Indy Reactions Chapter 3 CHAPTER 3 Optimization of Indy Reactions PREC PRE EE RCO LER ECE CRETE RRCOEERLEREREEEEOES 8 6 PRR EEE RERE REC ELE LE COCO LECCE L REISE EE ECO CER ECU CER EER EEE E CEC ERE EEC EOE ERE EERE ERLE General Notes e Indy is optimized to cycle 25 ul or 50 ul Inside this Chapter sample volumes TERANE e Unlike previous Idaho Technology instru Converting ments Bovine Serum Albumin BSA is RapidCycling not required for running samples on Indy protocols to Indy BSA is required only when using glass Converting Heat capillary tube
17. er set amplifies a region of the human Pyruvate Kinase gene The melting temperature for the forward and reverse primers are 61 C and 62 C respectively The product size is 291bp The cycling parameters optimized for this primer set is as follows 35 cycles of the following 1 Cycle Hold Time 35 Cycles Denaturation Annealing Extension Example Master Mix Contained Reagents Forward Primer KM Reverse Primer RS dNTPs MgCle Taq Polymerase TaqStart Antibody Template 2 00 9 92C s 0 sec 9 92 C s 0 sec 9 9 C s 17sec 9 9 C s 50 ul Reaction 10X Stock Final Concentrations 0 5uM 0 5uM 2mM each 0 2mM each 4 mM 0 8U 0 2ug 50ng ul 250ng ul total human genomic DNA The Taq TagStart mixture was obtained by combining 2ul of Taq 5 U l Roche Molecular Biochemicals with 2ul of TaqStart Antibody Clonetech and 21 ul of Taq Diluent Idaho Technology PCR is a proprietary technology owned by Roche Molecular Systems Inc RMS and have been sub licensed by PE Corporation PE in certain fields See disclaimer at front for more info 18 Setting up a 50 pl Reaction 5ul of combined 5uM forward and reverse primer 5ul of dNTPs 5ul 40mM MgCl2 5ul of Tag TaqStart mixture Sul of 50ng ul Human genomic DNA 25ul of water Note Divide each reagent by two for a 25 ul reaction Converting Heat Block Cycler Reactions to Indy Protocols developed using standard heat block cyclers are eas
18. ew user name select New User A screen will display to enter a user name using alphanumeric option Use the directional keypad to select a letter or number then press the Enter button When the name is completed use the keypad to select End The user name will be saved Select a user s name and press Enter The programs saved under that user will display To delete a user name or program select the desired user or program name and press 0 on the key pad The software will permanently delete the user or pro gram name Indy Air Thermocycler 29 Troubleshooting amp Maintenance Chapter 5 CHAPTER 5 Troubleshooting amp Maintenance LERET EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEELETEEETEEEEEEEETTETEEETETEEETETEEETETEETETEEEEEEEEEEEEEETETETETEETETEETETEEETETTETEEEETETEEECECEEET General Maintenance List Be sure to turn the power switch off A after use e Be sure the air intake underneath the machine is not obstructed Inspect the fan guard on the bottom of the instrument to Fuse Replacement ensure there is nothing blocking the air flow Inside this Chapter Troubleshooting amp A i e Keep lab bench and air intake of Indy free of dust Wipe the exterior surfaces i e top of sample tray lid vent with 10 bleach solution Chapter 5 Troubleshooting amp Maintenance 30 Indy Air Thermocycler Thermocouple The thermocouple regulates the tempera ture inside of the sample chamber a
19. icular purpose Idaho Technology shall not be liable for errors contained herein or for incidental consequential damages in connection with the furnishing performance or use of this material PCR is a proprietary technology covered by several US patents including US Patent Nos 4 683 195 4 683 202 and 4 965 188 and by issued and pend ing counterparts outside the U S These patents are owned by Roche Molecular Systems Inc RMS and have been sub licensed by PE Corporation PE in certain fields Depending on your specific application you may need a license from RMS or PE to practice PCR Additional information on pur chasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems 1145 Atlantic Avenue Alameda CA94501 or Applied Biosystems a k a PE 850 Lincoln Centre Drive Foster City CA94404 Customer and Technical Support Reach Us On the Web Idaho Technology s Web site is http www idahotech com We strongly encourage users to visit our Web site for answers to frequently asked questions additional Indy manuals parts and accessories Reach Us By E mail Contact Idaho Technology by e mail in the following areas Support idahotech com Technical Support it idahotech com General Support Reach Us By Phone Technical support is available during the following times 8 a m to 5 p m Mountain Standard Time For technical support call 1 800 735 6544 United States and
20. ily converted to Indy protocols Some reactions may require minor optimization Try the following steps 1 Decrease the annealing temperature by 3 to 5 C degrees Use a 0 second hold time 2 Eliminate or reduce the hold times for the denaturation step 3 Estimate the extension hold time by dividing the product by 25 base pairs and then adding 4 6 seconds In some cases the extra 4 6 sec onds may not be necessary however we recommend this step during the first optimization experiment The user may later decide to reduce the extension time once the reaction has been optimized right annealing temperature MgCl2 and primer concentration in order to decrease total reaction time If this is done the user needs to make sure that it does not interfere with efficiency of the reaction 4 Perform a magnesium buffer titration 1 5 mM final concentration Using a higher MgCl2 concentration may enhance the reaction 19 5 Boil DNA stock for about 10 minutes This should only be done once Optimizing A New Set of Primers To optimize a new primer set on Indy it is important to know the melting temperature Tm of each primer Idaho Technology recommends using a primer melting software i e Tm Tool to make calculations For free primer melting software download Hybridization Probe Tm Utility from Idaho Technology s Web site at www idahotech com Designing Amplification Protocol 1 Use a denaturation temperature of 94 C with a 0
21. intake and circulates the air through the outer chamber This air serves to cool the electron ic instrumentation housed in the outer chamber Inner Chamber Motor Bottom Air Intake Indy lowers sample tem LAs peratures by drawing air e through the side vents This air is then released through the top of the sample chamber A con sistent flow of air is creat ed to uniformly cool each sample Upper Air Intake amp Upper Fan PCR Sample Tubes Inner Chamber i nan i Outer Chamber l L Motor Bottom Air Intake amp Lower Fan During an Indy run heat ing elements inside the sample chamber heat the air Uniform air tempera ture is ensured when the lid is closed The circulat ing arrows in the image to the right show the air recirculation pattern Heated Lid PCR Sample Tubes Motor Bottom Air Intake Air Thermocycling There are several advantages to using air as the medium for heat exchange e Due to its low thermal mass air heats and cools much faster than other materials i e alloy heat block e Samples also heat and cool much faster when subjected to air ther mocycling e Faster temperature transitions or ramp rates allow samples to incu bate longer at their target temperature and spend less time heating and cooling Not only does this produce faster cycling times but it also reduces the amount of non specific product made Indy uses a softw
22. les By using air as an efficient method of heat transfer we have achieved faster reaction times compared to an alloy heat block cycler This allows Indy users to cycle up to 24 samples using standard 0 2 mL PCR tubes in less than 35 minutes In addition Indy s easy to use interface and small convenient design makes it the ideal instrument for any research institution In most thermocyclers the typical average temperature transition rate is approximately two degrees C per second A significant fraction of the cycle time is spent heating and cooling the sample as opposed to holding at opti mal temperatures Long reaction times of 2 6 hours are common and slow transition rates make it difficult to determine optimal temperatures and times for each stage of the cyclic reactions Indy combines the speed of air thermocycling with the convenience of using standard PCR tubes to hold samples Using heated air temperature transi tion rates of 5 10 C per second are easily obtained An internal fan mixes hot and cool air inside the chamber to provide temperature uniformity A complete 30 cycle reaction can be finished in as little as 35 minutes Indy is the fastest and smallest PCR tube thermocycler available today It can achieve total reaction times of 35 45 minutes which is less than half the time of standard cyclers Indy s products are easily visualized with ethid ium bromide stained agarose gels Additionally the rapid temperature cycli
23. lid and verify there is no debris in the chamber 34 Solution Make sure there is nothing on top of the sample tray that would obsiruct the venting system Anything higher than a sample container will cause a jittering noises when the vent tries to shut If the problem continues please contact our Technical Support at Idaho Technology Problem The machine appears to cycle normally but the reactions are not working Solution There are a number of factors that influence the outcome of reactions Review chapter 3 Optimization of Indy Reactions for reac tion suggestions If the problem continues please contact our Technical Support at Idaho Technology Problem The instrument attempts to perform thermocycling but the temperature display indicates that the chamber is not heating Cause and Solution The thermal fuse may be damaged To replace or fix the thermal fuse see chapter 5 Service and Maintenance in this manual for instructions If the problem continues please contact our Technical Support at Idaho Technology 35 Appendix Return amp Decontamination Forms lf you are returning a machine or product to Idaho Technology for replacement or repair fol low the basic guidelines for the decontamination and return your machine Once the forms have been filled out and the appropriate steps have been taken fax or e mail them to Idaho l Technology at fax 801 588 0507 or Inside this Chapter Support
24. lure to decontaminate before shipping to Idaho Technology Inc will result in the immediate return Decontamination Labels and affix of the unit at your expense Cut out label and attach it to shipping container L d QE aunyeuBIS SWEN JSWOISND GAVINY uonezuoulny Winey ABojouyoSs oyep Aoyjod uewdinby uinjay Jed paeuiwezuooap Useq sey JONPOJY siy QDIION uoneuiwezuovoq 41 PG 2 oF 2 DECONTAMINATION LABELS Please complete these one to the product and the other to the exterior of the shipping carton Failure to decontaminate before shipping to Idaho Technology Inc will result in the immediate return Decontamination Labels and affix of the unit at your expense Cut out label and attach it to shipping container ayeq ounyeuBIS ssaJppy JOWOISND SWEN JSWOISND GAVINY uogezuouny Winey ABojouyos oyepl Aoyjod juawudinby winjey Jed paeuiwezuooap usaq sey JONPOJY SIU QDIION uoneuiwezuovoq 42 Warranty amp Upgrades Warranty Idaho Technology Inc warrants the Indy Air Thermocycler against defects in material and workmanship for a period of one year from the date of pur chase If problems occur with your instrument a replacement machine will be shipped immediately via next day air In the event of machine failure please contact us at 1 800 735 6544 to arrange for the return repair and temporary replacement of your machine Upgrades Because of the mo
25. nd is a key part of Indy s precise temperature control The needle like thermocouple protrudes horizontally from the chamber wall halfway between the top and bottom of the cham ber at the 11 o clock position lf the thermocouple becomes noticeably bent while replacing the thermal fuse carefully bend it back to its original position If you notice difference in thermocycling please contact Idaho Technology Note Periodically inspect the thermocouple for damage Electric Fuse Replacement Tools amp supplies needed Flat blade screwdriver Replacement fuses 1 Turn off the power switch on the side of the instrument and unplug instrument Locate the fused switch on the right side of instrument 2 Insert a small flat bladed screwdriv er in fuse tray release slot and gen tly lift up This will allow the fuse tray to be removed 3 Replace fuses with appropriately rated fuses Fuse rating is located on the instrument tag next to the power switch Install fuse tray 4 Plug the Indy into a properly grounded power supply Turn switch back on and check for prop er operation of instrument If the problem continues please contact our Technical Support at Idaho Technology Thermal Fuse Replacement The heating coil on Indy has a thermal fuse designed to prevent damage to the instrument in the event of overheating If the instrument begins to over heat the thermal fuse will burn out and require replaceme
26. ng of this instrument can significantly enhance product specificity thus reducing primer dimers Indy Air Thermocycler Inside this Chapter Construction and Design Air Thermocycling 5 u About Indy Chapter 1 CHAPTER Indy was designed as a personal use thermocycler Its compact design and small foot print accommo dates any lab bench space The easy to use interface allows for single or up to 10 multiple users The sample chamber holds up to 24 samples using 0 2 ml PCR tubes A latch keeps the lid secure while Indy is running Indy is composed of two chambers an inner and outer chamber The inner chamber houses the sample wells heating elements and fan The outer chamber houses the electronic components Air is driven into the thermocycler by two fans upper and lower powered by a single fan motor When Indy is running the upper fan draws air from the side vents where it is heated by a heat coil The upper fan circulates air to accurately heat and cool samples To reduce condensation and help maintain chamber uniformity Indy has a second heater in the lid of the sample chamber This second heater is a cir cular circuit board with an embedded resistor mounted above the sample lids The heater is automatically turned on and maintained at 100 C when ever a thermocycling profile is run Outer Chamber The lower fan draws air from the bottom
27. nt If the instru ment is operated with a blown thermal fuse Indy will still attempt to execute the thermocycling program but the temperature display will show that the chamber is not heating Tools amp supplies needed Hex Wrench 1 16 Hex Wrench 0 050 Replacement Fuse 1 Turn off the power switch on the side of the instrument and unplug instrument Chapter 5 Troubleshooting amp Maintenance 32 Indy Air Thermocycler 2 Use the Hex Wrench 1 16 to remove the three screws holding the sample plate to the Indy body carefully remove the sample plate 3 Once the sample plate is removed use both hands and evenly remove the fan blade This will allow access to remove the thermal fuse 4 Using the 0 050 Hex Wrench care fully loosen the two screws holding the thermal fuse TWO SCREWS HOLDING THE THER A DO NOT COMPLETELY REMOVE THE MAL FUSE LOOSEN ONLY 5 With the two screws loosened carefully slide the fuse out from under the two screws and lift the fuse out of the sample chamber body 6 Carefully replace the fuse by sliding the new fuse under the two screws Aga Once the fuse is securely fastened SN ine aN w stan G n under the screws tighten using the 0 050 Hex Wrench 7 Replace the sample plate to its original position and secure it to Indy s body with the three screws 8 Plug the Indy into a properly grounded power supply Turn the power switch on and check for proper o
28. peration of Indy If the problem con tinues please contact our Technical Support at Idaho Technology 33 Indy Troubleshooting Problem The Indy screen goes blank during a run Cause and Solution This may be caused by a static discharge to the chassis of the instrument Even though the screen has gone blank the machine will continue temperature cycling correctly Allow the machine to finish the program turn the power switch off for three seconds and then turn the switch back on If the problem continues please contact our Technical Support at Idaho Technology Problem The machine will not turn on Solution Verify the proper outlet power and the power cord connec tion Solution Check the power fuses in the fuse tray located next to the power switch to see if they need to be replaced If the fuse is dam aged see chapter 5 Service and Maintenance in this manual for replacement instructions If the problem continues please contact our Technical Support at Idaho Technology Problem The machine will not run when the run function is selected Cause and Solution The Chamber Lid may not be closed and latched securely Press firmly on the latch handle to secure it to the body If the problem continues please contact our Technical Support at Idaho Technology Problem There is an unusual noise coming from the machine when it is cycling Solution Turn the power off disconnect the power cord lift the
29. s Block protocols to Indy e Samples run on Indy do not require an oil overlay Indy s sealed chamber and heat ed lid maintains uniform temperatures with minimal sample condensation A small amount of condensation may result on the side of the tubes Simply cen trifuge the sample tube before opening to prevent contamination Optimizing a New Primer Set 16 Converting RapidCycling protocols to Indy Standard PCR tubes offer some advantages over capillary tubes First sample handling is simpler when using PCR tubes Glass capillaries are fragile and require heat sealing Second standard master mixes can be used without the addition of BSA In general RapidCycling protocols transfer directly to Indy The user may need to scale up the reaction volume from 10 ul to 25 or 50 ul When run ning RapidCycling reactions on Indy adding BSA Bovine Serum Albumin to the master mix is unnecessary since glass capillaries are not used If the reactions do not work properly on Indy try the following 1 Increase the annealing temperature 2 Reduce the hold times in the annealing and extension steps 3 Perform a magnesium buffer titration 1 5 mM final concentration Using a higher Mg concentration may enhance the reaction NOTE BSA is not required to run Indy reactions 17 Sample Reaction and Master Mix Amplification Conditions for the PyrvK F PyrvK R forward and reverse Primer set The PyrvK F and PyrvK R prim
30. second hold time 2 Select an annealing temperature 5 C lower than the primer with the lowest melting temperature Tm Use a 0 second hold time 3 Use 72 C as the extension temperature Estimate the extension hold time by dividing the product by 25 base pairs and then adding 4 6 seconds In some cases the extra 4 6 seconds may not be neces sary however we recommend this step during the first optimization experiment The user may later decide to reduce the extension time once the reaction has been optimized right annealing temperature MgCl2 and primer concentration in order to decrease total reaction time If this is done the user needs to make sure that It does not interfere with efficiency of the reaction 4 Perform a magnesium buffer titration 1 5 mM final concentration 20 Inside this Chapter Quick Run Copy Programs Adding New Users Adding New Programs 21 Programming Indy Chapter 4 CHAPTER Programming Indy Overview of Indy Functions When the user turns Indy on an introductory screen containing the software version will briefly display Next the main menu listing Indy func tions will display Indy has two main functions Quick Run Select User 22 To enter a function use the directional keypad to select the desired function and then press the Enter button To return to the previous menu press the Esc button Quick Run This function can be used to conduct a basic
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