DNA 12K Assay User Guide
Contents
1. Up to 96 samples in a 96 well or 384 well plate can be run with a single chip preparation DNA 12K Assay User Guide Chip Well Aspiration Using a Vacuum 29 Chip Well Aspiration Using a Vacuum Aspirating with a pipette can leave used reagents in the chip wells For this reason PerkinElmer recommends vacuuming the wells instead This can be accomplished by attaching a permanent pipette tip to a house vacuum line with trap Figure 20 To avoid contamination use a new disposable pipette tip over the permanent tip for each chip aspirated Figure 21 Figure 20 A Permanent pipette tip attached to a house vacuum line B vacuum line with trap Figure 21 Replacing the disposable pipette tip PN CLS140167 Rev C DNA 12K Assay User Guide Customer Technical Support 30 Customer Technical Support PerkinElmer Inc 68 Elm Street Hopkinton MA 01748 1668 PerkinElmer Technical Support Phone USA Toll Free 800 762 4000 Phone Worldwide 1 203 925 4602 Fax 1 203 925 4602 Email global techsupport perkinelmer com Internet www perkinelmer com For additional assay and instrument troubleshooting refer to the LabChip GX Touch Software Help file PN CLS140167 Rev C DNA 12K Assay User Guide Licenses and Rights of Use 31 Licenses and Rights of Use Label Licenses This product is provided under an intellectual property license from Life Technologies Corporation The purchase of this product conveys to th2. Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 12 Wash screen 6 Remove the chip from the instrument and place it in the plastic storage container Add an additional amount of Storage Buffer to well 1 7 Cover the wells with Parafilm to prevent evaporation and store at 4 C Storage of a chip with dry wells may cause it to become clogged PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 16 Chip Cartridge Cleaning 1 Daily a Inspect the inside of the chip cartridge and O rings for debris b Use the provided lint free swab dampened with water Milli Q or equivalent to clean the O rings using a circular motion If the O rings stick to the chip or a pressure leak is detected perform the more extensive monthly cleaning procedure 2 Monthly PN CLS140167 Rev C a To reduce pressure leaks at the chip interface clean the O rings frequently Remove the O rings from the top plate of the chip interface on the LabChip GX Touch GXIl instrument Soak O rings in water Milli Q9 or equivalent for a few minutes Clean the O ring faces by rubbing between two fingers Wear gloves To reduce the occurrence of current leaks clean the chip interface frequently Clean the top plate of the chip interface using the provided lint free swab dampened with water Milli Q or equivalent Allow the O rings and chip interface to air dry Reinsert the O ri
3. Figure 3 Note The marker well may need to be replenished if the chip is in idle mode on the instrument for an extended period of time 7 Make sure the rims of the chip wells are clean and dry 8 IMPORTANT Ensure chip well 1 waste well is empty before placing the chip into the instrument Inserting a Chip into the LabChip GX Touch GXII Touch Instrument 1 Check that the sample plate Buffer Tube and Ladder Tube are properly placed on the instrument 2 Remove the chip from the chip storage container and inspect the chip window Clean BOTH sides of the chip window with the PerkinElmer supplied clean room cloth dampened with a 70 isopropanol solution in DI water 3 Touch the Unload Chip button on the Home screen Chip Status Type HT DNA 12K Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 2000 Samples Left Status Figure 4 Home screen 4 Insert the chip into the LabChip GX Touch GXII Touch instrument Figure 5 and close the chip door securely PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 11 Figure 5 Chip in the LabChip GX Touch GXII Touch instrument 5 Touch the Load Plate button on the Home screen Figure 4 to retract the sample plate and send the sipper to the Buffer Tube Note Do not keep the chip door open for any length of time Dye is sensitive to light and can be photobleached 6 The Assay Choice window will appear Figure 6 Touch the desired assay and
4. a Expected Peak File m Excluded Peak File Figure 10 Run setup screen 5 Touch Start to begin the run m Read Plate Barcode m Default borg Default LabChip GX I Touch HT DNA Quantitative PN CLS140167 Rev C Run Select Wells Setup Run Start Run Chip Status Type HT DNA 12K Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 2000 Samples Left Run Parameters Operator IM Assay HT DNA 12K Extended Time Ver2 Data Path C Program Files x86 PerkinElmer LabChip GX Touch Data Plate Name BioRad 96 HSP 96xx Sip 4 mm Barcode N A Plate Cycles 1 Auto Export No Sample Saver No Random Selection No Figure 11 Starting a run DNA 12K Assay User Guide Preparation Procedures 15 Storing the Chip After use the chip must be cleaned and stored in the chip container The procedure below can be conducted the following day when running overnight 1 Place the chip into the plastic storage container The sipper should be submerged in the fluid reservoir 2 Remove the reagents from each well of the chip using vacuum 3 Each active well 1 3 4 7 8 and 10 should be rinsed and aspirated twice with water Milli Q or equivalent 4 Add 120 uL of Storage Buffer white cap to the active wells 5 Place the chip in the LabChip GX Touch GXII Touch instrument and touch the Wash button in the upper right corner in the Home Screen R Chip Status Type HT DNA 12K
5. then touch OK This chip supports multiple assays Select one HT DNA High Sens HT DNA 1K HT DNA 12K HT gDNA Figure 6 Assay Choice menu Notes The chip may be run with multiple assays but only one assay type should be run on the chip Be sure to periodically clean the O rings on the top plate of the chip interface on the LabChip GX Touch GXIl Touch Use the provided lint free swab dampened with water to clean the O rings using a circular motion Allow the O rings to dry before inserting a chip PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 12 Running the Assay Note Chips can be primed independently from running assays Touch the Prime button on the Home screen Make sure the Buffer Tube is placed on the instrument rro iLAALA Change Assay Folder Chip Status Assay Folder Type HT DNA 12K C Program Files x86 PerkinElmer LabChip GX Touch Assay Chip Reagents N A Assay eee i a IO ChipiExpiration Jin 2020 HT DNA 12K Extended Time Ver2 M Chip Life 2000 Samples Left Run Test Ladder after Prime Buffer Prime Figure 7 Chip priming screen 1 Touch the Run button see Figure 7 2 Select the appropriate assay type see m For DNA 12K assays the appropriate assay types are DNA 12K For sizing of DNA fragments in the 100 to 12000 bp range DNA 12K Extended Time To be used only if peaks are cut off using the standard DNA 12K script occurs in some high salt s
6. 0X DNA 12K Ladder Yellow 1 vial 0 15 mL DNA 12K Marker Green vial 1 5 mL PN CLS140167 Rev C DNA 12K Assay User Guide Specifications Table 4 Consumable Items 5 Item Supplier and Catalog Number Quantity Spin Filters Costar Cat 8160 8 Centrifuge Tubes Eppendorf Cat 4 022363352 5 2 0 mL Ladder Tubes Genemate Cat C 3258 1 10 0 2 mL Detection Window VWR Cat 21912 046 1 Cleaning Cloth Swab ITW Texwipe Cat TX758B 3 Buffer Tubes E amp K Scientific Cat 697075 10 0 75 mL NC Table 5 DNA LabChips Item Catalog Number with GX Touch GXII Touch HT DNA Extended Range Chip 12K for use Cat 760517 with GX Touch GXII Touch 24 DNA Extended Range Chip 12K for use Cat CLS138948 PN CLS140167 Rev C DNA 12K Assay User Guide Safety Warnings and Precautions 6 Safety Warnings and Precautions WARNING A For Research Use Only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals CAUTION We recommend that this product and components be handled only by those who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing such a
7. O rings on the top surface of the chip interface and clean if necessary PN CLS140167 Rev C DNA 12K Assay User Guide LabChip Kit Essential Practices 23 LabChip Kit Essential Practices General PN CLS140167 Rev C To ensure proper assay performance please follow the important handling practices described below Failure to observe these guidelines may void the LabChip Kit product warranty Note It is important to keep particulates out of the chip wells channels and capillary Many of the following guidelines are designed to keep the chips particulate free For assay and instrument troubleshooting refer to the LabChip GX Touch software Help file or call PerkinElmer Technical Support at 1 800 762 4000 Allow the chip sample plate and all reagents to equilibrate to room temperature before use approximately 20 to 30 minutes Clean the O rings in the chip interface weekly and the electrodes daily Refer to the Instrument Users Guide Maintenance and Service section for procedures Avoid use of powdered gloves Use only non powdered gloves when handling chips reagents sample plates and when cleaning the instrument electrodes and electrode block Calibrate laboratory pipettes regularly to ensure proper reagent dispensing Only the PerkinElmer supplied clean room cloth can be used on the chip to clean the detection window Water used for chip preparation procedures must be 18 megohm 0 22 um filtered water Milli Q
8. RK ONE RANKAR DE eR 17 Troubleshooting Li diode neca amu ax deano ta cC Ceux Fo oon 2 cuAda pu Ada ENN ERLBAS AM a SUAE CERERI YSR 18 LabChip Kit Essential PraCtiCeS sssssssssssssssssssssrnrnsnsrrnnnnnn nn nn enne nnn 23 Cr pe E MO en 23 Reagents n Eda 24 ei ENE ht Ho ee 25 Samples MER EI T III M M 28 Chip Well Aspiration Using a Vacuum eene nennen nennen nnn nnn 29 Customer Technical Support eee erinnert antt inni e ne AREAN cu mnnn 30 Licenses and Rights oT USO icone rr o nea S ii aic ie a e rr ora an 31 PN CLS140167 Rev C DNA 12K Assay User Guide Specifications 3 Specifications Assay Specifications Table 1 Assay Specifications Sizing Range 100 12000 bp Sizing Resolution 1096 from 150 1000 bp 1596 from 1000 2000 bp 2096 from 2000 8000 bp 2596 from 100 150 bp 8000 12000 bp Sizing Accuracy t 1096 Sizing Precision 596 CV Linear 0 25 50 ng uL per fragment Concentration Range Sensitivity 0 25 ng uL Maximum Total DNA 60 ng uL total 50 ng uL per fragment Concentration Quantitation 40 or 1 ng uL whichever is greater Accuracy Quantitation 2096 CV from 100 5000 bp 25 CV from Precision 5000 12000 bp Number of Samples 400 samples four 96 well plates or one per Chip Prep 384 well plate a
9. Resolution is defined as half height or better separation of two peaks Actual Separation performance can depend on the sample and application Peaks that are resolved less than half height can still be accurately identified by the system software PN CLS140167 Rev C DNA 12K Assay User Guide Sample Conditions Specifications 4 Table 2 Sample Conditions Additives PerkinElmer recommends that BSA and detergents exceeding 0 05 mg mL and 0 01 v v respectively in concentration not be used Higher concentrations can result in chip failure In addition non aqueous solvents are not compatible with DNA LabChip protocols Particulates Salt Concentration All sample plates should be spun down prior to analysis All buffers should be filtered with a 0 22 um cellulose acetate filter Total salt concentration must not exceed 125 mM Plasmids Plasmid concentration in samples must be below 20 ng uL Please note that although the DNA Assays cannot analyze plasmids the presence of plasmids above 20 ng uL can interfere with assay results Kit Contents Storage When not in use store chips and reagents refrigerated at 4 C Do not leave chips and reagents unrefrigerated overnight Table 3 Reagent Kit Contents PN 760569 Reagent Vial Quantity DNA Dye Concentrate Blue Q 1 vial 0 08 mL Chip Storage Buffer White 5 vials 1 8 mL each DNA 12K Gel Matrix Red Q 3vials 1 6 mL each 1
10. User Guide Chips Repriming Chips LabChip Kit Essential Practices 25 Note Buffer tubes filled with 1X DNA sample buffer or water should be placed into the instrument while priming or washing chips Touch the Unload Chip button on the Home screen to open the instrument door Place the chip into the instrument Close the chip door securely and choose the corresponding assay Touch the Prime button on the Home screen to reprime the chip Washing and Repriming Chips PN CLS140167 Rev C Touch the Unload Chip button on the Home screen to open the instrument door Return the chip to the chip container ensuring the sipper is immersed in fluid Thoroughly aspirate all fluid from the chip wells using a vacuum line Ensure that each active well 1 3 4 7 8 and 10 is rinsed and completely aspirated twice with water Milli Q or equivalent Do not allow active wells to remain dry Add 120 uL of Storage Buffer to each active well 1 3 4 7 8 and 10 Place the chip in the LabChip GX Touch GXIl Touch instrument Close the chip door securely Touch the Wash button on the Home screen Figure 18 DNA 12K Assay User Guide LabChip Kit Essential Practices 26 Chip Status Type HT DNA 12K Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 18 Wash screen After the completion of the wash cycle return the chip to the chip container ensuring the sipper is immer
11. ample buffers HT DNA 12K Extended Time HT DNA 12K Extended Time HT DNA 12K Figure 8 Assay Choice window 3 Select the plate name well pattern and whether to read wells in columns or rows Select number of times each well is sampled under Adv Settings Figure 9 Touch the green arrow button PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 13 Run Select Wells Setup Run Start Run Assay Folder CAProgram Files x86 PerkinElmer LabChip GX Touch Assay Select Assay Select Plate Type 3 G z HT DNA 12K Extended Time Ver2 BioRad 96 HSP 96xx Sip 4 mm Figure 9 Selecting wells 4 Inthe Setup Run tab select the operator name the option to read barcode the destination of the file the inclusion of sample names expected peaks and excluded peaks and the filename convention Select Auto Export to export results tables automatically Figure 10 Touch the green arrow button PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 14 LabChip GX II Touch HT DNA Quantitative Run Select Wells Setup Run Start Run Operator IM Data Path C Program Files x86 PerkinElmer LabChip GX Touch Data Iv Copy to C Program Files x86 PerkinElmer LabChip GX Touch DataCopy Iv Create Daily Sub Directory m Auto Export Data File Name Labchip 2014 06 09 01 26 07 9xd File Prefix Project name F Computer Name C Barcode Iv Date Advanced Settings m Sample Names File
12. ations of the Buffer Tube and Ladder Tube in the GXII Touch instrument Preparing the Chip 1 PN CLS140167 Rev C Allow the chip to come to room temperature DNA 12K Assay User Guide Preparation Procedures 9 2 Use a pipette tip attached to a vacuum line to thoroughly aspirate all fluid from the chip wells see Figure 2 For more details on how to set up a vacuum line see page 29 Figure 2 Using a vacuum to aspirate the chip wells is more effective than using a pipette 3 Rinse and completely aspirate each active chip well 1 3 4 7 8 and 10 twice with water Milli Q9 or equivalent Do not allow active wells to remain dry 4 f any water spills onto the top and bottom chip surfaces during rinsing aspirate using the vacuum line DO NOT run the tip over the central region of the detection window Use the provided Detection Window Cleaning Cloth dampened in water Milli Q9 or equivalent or alcohol to clean chip detection window as needed 5 Using a reverse pipetting technique add 75 uL Gel Dye solution to chip wells 3 7 and 8 and 120 uL to well 10 as shown in Figure 3 DNA O C e CO 09 Marker Gel Dye Figure 3 Reagent placement PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 10 6 Add DNA Marker green cap to chip well 4 Use 50 uL DNA Marker for a 96 well plate and 120 uL DNA Marker for a 384 well plate or multiple 96 well plates as shown in
13. e buyer the non transferable right to use the data for internal research and training purposes solely in combination with PerkinElmer instrumentation and not to generate revenue which may include but is not limited to use of the product 1 in manufacturing or in quality assurance or quality control of a commercial product 2 to provide a service information or data for a fee or other consideration 3 for therapeutic or prophylactic purposes 4 for diagnostic use and 5 for resale whether or not such items are resold for use in research For information on purchasing a license to this product for any other purposes contact Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 USA or outlicensing lifetech com Rights of Use The chip and reagents supplied with this kit are sold with limited rights of use The chip may only be used with the specific quantity of reagents supplied with this kit The purchaser has no right or license to refurbish reuse remanufacture or otherwise use the chip with any other reagents than those specifically supplied in this kit For more information on the terms and conditions of use of these chips and reagents please read your LabChip GX Touch User Guide and refer to the applicable label license The reagent kit contains materials that are provided under a license from Life Technologies Corporation for research use only PerkinElmer LabChip and the LabChip logo are registered trademarks
14. e volume in the well of the plate 2 If the missing sample peaks occurred only in a few wells of the plate check those wells for air bubbles 3 The sipper is not reaching the sample due to an incorrect capillary height setting or incorrect plate definition 4 If the plate has been uncovered for some time sample evaporation might have occurred 5 Debris from the sample or sample prep is clogging the sipper What to do 1 Add more sample to the well PN CLS140167 Rev C DNA 12K Assay User Guide Troubleshooting 19 Manually insert a larger volume pipette tip 7100 pL into the sample well and dislodge the bubble Rerun these sample wells Check the plate definitions Check the sample wells especially around the edge of the plate where evaporation is fastest and make a fresh plate if volumes are low If you suspect there may be debris in your samples spin the sample plate down in a centrifuge e g 3000 rcf for 5 minutes Unclog the sipper by repriming the chip See LabChip Kit Essential Practices on page 23 for instructions on how to reprime the chip Symptom No ladder peaks but sample peaks and marker peaks are present Possible causes 1 Low or no ladder volume in the Ladder Tube What to do 1 Add more ladder to the Ladder Tube and restart the run Recommended standard ladder volume is 120 uL minimum volume is 100 uL Symptom No marker peaks but sample peaks are present Possible causes 1 No
15. j gt Perkin DNA 12K Assay User Guide For LabChip GX Touch GXIl Touch ce Copyright 2014 PerkinElmer Inc All rights reserved PerkinElmer is a registered trademark of PerkinElmer Inc All other trademarks are the property of their respective owners PN CLS140167 Rev C Contents SPECIN CANON ST unione ianua NE E hs 3 Assay Specificati nS x eios ot D d RE DINE ot tu dp PS Ric odeurs 3 Sample Conditlons ester ee trt e ee holes Dee vel uat ua uiua De e ER Det d 4 Kit Contents P n c ii 4 Safety Warnings and PreCaUtiONS sssssssssrsssrsssssrrrsssnsrrrrrnnn rr nennen nene 6 Preparation Procedures ias noie enc tana etd noe Han n rk xerit ce s R ARENA RER KRK nn nn 7 Additional Items Required 5 rodeo Ei at eee Cedere tea beni ate DR Krona 7 Preparing the Gel Dye Solution eseeeesseeeeeeeeeeenenen men 7 Preparing the DNA Samples and DNA Ladder ismsssssrrrresssrrrresrrrrrerrrnrrrrrrnnr rr rerna na 8 Preparing the Butter Tube zs re Deeds Dune dvi KRK eee 8 Preparing the Chip tT 8 Inserting a Chip into the LabChip GX Touch GXII Touch Instrument 10 RUNNING Me ASSAY xix c M n 12 SLOUGH Te jose RM DIL DEED 15 Chip Cartridge Cleaning ees iuret reb hr eue Ye EX ERR ERROR UE RR KK E gea aed 16 FRO SUS ns oss d ran i eiie ced vieni oni i ceni RH Fo EE Rd EU c FRE ub ER ARE uu aw E REG 17 DNA 12K Ladder Result rsisi reper ete RC ERR S
16. marker added to chip well 4 2 If there is marker solution in chip well 4 the problem may be due to a marker channel clog What to do 1 This may be due to not filling marker well or chip remaining idle PN CLS140167 Rev C on instrument for extended period of time Add or replenish the marker solution in the chip using the following procedure e Touch the Unload Chip button on the Home screen to open the chip door Return the chip to the chip container ensuring the sipper is immersed in fluid e Thoroughly aspirate all fluid from chip well 4 using a vacuum line Ensure that chip well 4 is rinsed and completely aspirated twice with water Milli Q9 or equivalent Add Marker Solution green cap to chip well 4 DNA 12K Assay User Guide Troubleshooting 20 Reinsert the chip back into the instrument Restart the run 2 Perform a marker channel unclogging procedure by repriming the chip See LabChip Kit Essential Practices on page 23 for instructions on how to reprime the chip Symptom Ladder traces show up in the lanes following the ladders delayed sip Y Abgoed Time sec Figure 14 Small ladder peaks in sample well caused by delayed sip Possible causes 1 Separation channel overloaded with sample 2 Partial clog in the separation channel What to do 1 Lower the starting sample concentration 2 Reprime the chip See LabChip Kit Essential Practices on page 23 for instruc
17. ngs into the chip cartridge DNA 12K Assay User Guide Results 17 Results DNA 12K Ladder Result The electropherogram of a typical DNA 12K ladder is shown in Figure 13 Between the upper and lower markers peaks in order of increasing migration time correspond to ladder fragments of 100 300 500 700 1100 1900 2900 4900 7000 and 10000 bp Lower Marker sot Upper 60 Marker e mo o 40 3 Hm j 20 i od D 20 25 40 45 ai TANE Figure 13 DNA 12K ladder electropherogram PN CLS140167 Rev C DNA 12K Assay User Guide Troubleshooting 18 Troubleshooting Note Some of the data examples shown in this section were generated with assays other than the assay described in this user guide Symptom No ladder or sample peaks but marker peaks detected Note The lower marker peak height will most likely be greater than normal height Possible causes 1 Air bubble in sipper introduced during chip priming What to do 1 Reprime the chip See LabChip Kit Essential Practices on page 23 for instructions on how to reprime the chip Symptom Missing sample ladder and marker peaks Possible causes 1 Clog in sipper or marker channel of chip What to do 1 Reprime the chip See LabChip Kit Essential Practices on page 23 for instructions on how to reprime the chip Symptom Ladder detected but no sample peaks Possible causes 1 The sipper is not reaching the sample due to low sampl
18. of PerkinElmer Inc and or its parent affiliates and or subsidiary companies collectively PerkinElmer The PerkinElmer logo is a registered trademark of PerkinElmer Inc All other trademarks and registered trademarks are the property of their respective holders 2014 PerkinElmer Inc http www perkinelmer com PN CLS140167 Rev C DNA 12K Assay User Guide j gt Perkin PerkinElmer Inc 68 Elm Street Hopkinton Massachusetts 01748 U S A TEL 508 435 9500 FAX 508 435 3439 http www perkinelmer com
19. or equivalent Using the Reverse Pipetting Technique described next will help avoid introducing bubbles into the chip when pipetting the gel PerkinElmer warrants that the LabChip Kit meets specification at the time of shipment and is free from defects in material and workmanship LabChip Kits are warranted for 90 days from the date of shipment All claims under this warranty must be made within thirty days of the discovery of the defect DNA 12K Assay User Guide LabChip Kit Essential Practices 24 Reverse Pipetting Technique Reagents PN CLS140167 Rev C STEP 1 STEP 2 STEP 3 STEP 4 Figure 17 Reverse pipetting Depress the pipette plunger to the second stop Aspirate the selected volume plus an excess amount from the tube Dispense the selected volume into the corner of the well by depressing plunger to the first stop Withdraw the pipette from the well Store reagents at 4 C when not in use The LabChip dye contains DMSO and should be thawed completely before use It is recommended that you prepare aliquots to reduce the time required for thawing Gently vortex all kit reagents before use Dispense reagents into chip wells slowly without introducing air bubbles Insert the pipette tip vertically and to the bottom of the chip well Protect the dye and Gel Dye mixture from light Store in the dark at 4 C when not in use The Gel Dye mixture expires 3 weeks after preparation DNA 12K Assay
20. ored refrigerated prior to first use Cover the active wells on the chip with Parafilm9 and store at 4 C If using the chip again within 24 hours it may be left at room temperature Do not allow the liquid in the chip container to freeze as this may lead to poor chip performance Do not submerge the chip in any solution The entire chip surface must be thoroughly dry before use The sipper must be kept immersed in fluid at all times and should not be exposed to an open environment for long periods of time Use care in chip handling to prevent sipper damage Damage to the sipper can result in inconsistent sampling Avoid exposing the chips to dust by keeping them in a closed environment such as in the chip container or in the instrument before and after chip preparation DNA 12K Assay User Guide Samples PN CLS140167 Rev C LabChip Kit Essential Practices 28 Chips can be prepared and left idle on the instrument for up to 8 hours This workflow allows analysis of samples as needed throughout the day without having to re prep the chip as long as the maximum number of samples per chip prep is not exceeded PerkinElmer recommends the chip be re prepared after it has been idle for 8 hours Prepared sample plates should be free of gas bubbles and particulate debris both of which may inhibit sipper flow Sample plates containing gas bubbles and or particulate debris should be spun down at 3000 rpm 1250 rcf prior to analysis
21. pected Note Some migration time variance between chips or within a plate is considered normal chip performance All chips are QC tested at PerkinElmer prior to shipment Possible causes 1 Incorrect Gel Dye ratio Migration time is sensitive to dye concentration and peaks will migrate too fast or too slow if the dye concentration in the gel is too low or too high respectively Note Excess dye in the separation channel will slow down migration and less dye in the separation channel will make peaks migrate faster 2 Particulates from the samples may be clogging the separation channel this will slow down migration 3 Gel Dye was not primed properly into the chip What to do 1 Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture See LabChip Kit Essential Practices on page 23 for instructions on how to wash and reprime the chip 2 If fast or slow migration is observed repeatedly on a new chip contact technical support to arrange return of the chip to PerkinElmer Please send a data file showing the failure along with the return request 3 Minimize the loading of particulates in the sample by performing a centrifuge spin of the sample plate e g 3000 rcf for 5 minutes before starting a new run The debris may be flushed out of the chip by washing and re priming the chip See LabChip Kit Essential Practices on page 23 for instructions on how to wash and reprime the chip 4 Check the
22. s approximately 550 uL each Centrifuge at 9300 rcf for 7 5 minutes at room temperature Discard filters label and date the tubes Store in the dark at 4 C Use within 3 weeks DNA 12K Assay User Guide Preparation Procedures 8 Preparing the DNA Samples and DNA Ladder Notes DNA Ladder should be prepared in the same buffer as your DNA samples A buffer mismatch between sample and ladder may lead to inaccurate quantitation and sizing DNA sample buffer solution is the user s DNA buffer such as PCR buffer etc 1 In the provided 0 2 mL Ladder Tube add 12 uL of DNA Ladder yellow cap to 108 uL of 1X DNA sample buffer solution Mix thoroughly by pipetting the solution up and down a few times Avoid creating air bubbles Ensure there are no air bubbles in the Ladder Tube Insert the Ladder Tube into the ladder slot on the LabChip GX Touch GXII Touch instrument Recommended sample volumes are 25 uL for a 384 well plate or 40 uL for a 96 well plate Preparing the Buffer Tube 1 Sample 0000000009 Add 750 uL of 1X DNA Sample Buffer solution to the 0 75 mL Buffer Tube provided with the reagent kit Ensure there are no air bubbles in the Buffer Tube Insert the Buffer Tube into the buffer slot on the LabChip GX Touch GXIl Touch instrument Buffer Tube Ladder Tube uu AAI FF I YS Well A1 000000700797 RP P PP P UL d Ladder AWAY NI N NI IYI YI YH YH SY Tube Buffer Tube Figure 1 Loc
23. s laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In case of contact with skin or eyes wash immediately with water WARNING AA Dye Concentrate contains DMSO 824 25 Avoid contact with skin and eyes PN CLS140167 Rev C DNA 12K Assay User Guide Preparation Procedures 7 Preparation Procedures Additional Items Required 18 megohm 0 22 um filtered water Milli Q or equivalent 7096 isopropanol solution in DI water Bio Rad Hard Shell 384 well Skirted PCR Plates Cat HSP 38XX recommended PerkinElmer Hard Shell thin wall 96 well skirted PCR plate blue Cat 6008870 recommended Note Allow the chip and reagents to equilibrate to room temperature for 20 30 minutes before use Preparing the Gel Dye Solution Notes The Dye Concentrate contains DMSO and must be thawed completely before use The dye is light sensitive Do not expose Dye or Gel Dye to light for any length of time Keep prepared Gel Dye in the dark 1 PN CLS140167 Rev C Vortex the thawed DNA Dye Concentrate blue cap for 10 15 seconds before use Transfer 1 0 mL of DNA Gel Matrix red cap using a reverse pipetting technique to the centrifuge tube provided with the reagent kit Add 12 5 uL of DNA Dye Concentrate blue cap Q9 to the centrifuge tube Vortex the solution until it is well mixed and spin down for a few seconds Transfer the mixture into two spin filter
24. sed in fluid e Thoroughly aspirate all fluid from the chip wells using a vacuum line Ensure that each active well 1 3 4 7 8 and 10 is rinsed and completely aspirated twice with water Milli Q or equivalent Do not allow active wells to remain dry Add Gel Dye solution to chip wells 3 7 8 and 10 using a Reverse Pipetting Technique as shown in Figure 17 e Add Marker green cap to chip well 4 Use 50 uL for 96 well or 120 uL for 384 well plate analysis Please note that the marker well may need to be replenished if the chip is in idle mode on the instrument for an extended period of time e Place the chip into the LabChip GX Touch GXII Touch instrument Close the chip door securely Touch the Run or Prime button on the Home screen PN CLS140167 Rev C DNA 12K Assay User Guide LabChip Kit Essential Practices 27 If air bubbles are not dislodged after a reprime apply a vacuum to the sipper Perform this by filling all active wells with 100 uL of Chip Storage Buffer Then suction the sipper with a vacuum line as shown in Figure 19 until droplets of fluid flow out from the sipper When suctioning the sipper be careful not to bend or break the sipper To facilitate this cut the end of the pipette tip attached to the vacuum line to widen the mouth Figure 19 Removing an air bubble or clog by suctioning the sipper with a vacuum line Other Considerations PN CLS140167 Rev C DNA chips should be st
25. tions on how to reprime the chip PN CLS140167 Rev C DNA 12K Assay User Guide Troubleshooting 21 Symptom Unexpected sharp peaks 600 Lower Upper marker marker Peak caused by particulates Fluorescence w e o 40 45 50 55 Aligned Time sec Figure 15 Unexpected sharp peak Possible causes Dust or other particulates introduced through sample or reagents What to do 1 Doone or all of the following Replace the 18 megohm 0 22 Lm filtered water Milli Q or equivalent water used for chip preparation Replace the buffer used for sample and reagent preparation Use a0 22 micron filter for all water and buffers used for chip sample and reagent preparation Spin down sample plate to pellet any particulates Symptom Humps in several electropherograms which do not correspond to sample data 1250 1000 o o 500 Fluorescence 20 25 30 35 40 45 50 55 60 Time seconds Figure 16 Humps in several electropherograms PN CLS140167 Rev C DNA 12K Assay User Guide Troubleshooting 22 Possible causes 1 Electrode 7 is dirty and has contaminated the Gel Dye mixture in well 7 What to do 1 Before restarting the run clean electrode 7 Remove the chip and follow the electrode cleaning procedure We recommend using the provided swab and isopropanol to manually clean electrode 7 Symptom Peaks migrating much faster or slower than ex
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