DNA High Sensitivity Assay User Guide
Contents
1. 5 from 100 500 bp 10 from 50 100 bp 500 1000 bp 15 from 1000 3000 bp 22 from 3000 5000 bp Sizing Accuracy 10 Sizing Precision 5 CV Linear Standard Sample Workflow Concentration 10 pg uL 500 pg uL per fragment from 50 Range bp to 2000 bp 50 pg uL 500 pg uL per fragment from 2000 bp to 5000 bp 100 pg uL 5 ng uL for smears Limited Sample Workflow initial concentration 20 pg uL 500 pg uL per fragment from 50 bp to 2000 bp 100 pg uL 500 pg uL per fragment from 2000 bp to 5000 bp 200 pg uL 5 ng uL for smears Standard Workflow 5 pg uL per fragment 100 pg uL for smears Limited Sample Workflow initial concentration 10 pg uL per fragment 200 pg uL for smears Maximum Total DNA 5 ng uL total 500 pg uL per fragment Concentration Quantitation 30 Accuracy 1 All specifications pertaining to DNA fragments were determined using ladder as sample in TE buffer All specifications pertaining to DNA smears were determined using Covaris sheared control genomic DNA human male in TE buffer Shearing time was 30s or 240s PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Specifications 4 Table 1 Assay Specifications Continued Sizing Range 50 5000 bp Quantitation 20 CV Precision Analysis Time 68 seconds per sample 2 5 hours for 96 samples Number of Samples per Chip Prep 96 samples a Resolutio2. Table 3 Reagent Kit Contents PN CLS760672 Reagent Vial Quantity DNA Dye Concentrate Blue 1 vial 0 09 mL Chip Storage Buffer White 9 vials 1 8 mL each DNA HiSens Gel Matrix Red 5 vials 1 1 mL each O e 10X DNA HiSens Ladder Yellow 1 vial 0 26 mL DNA HiSens Marker Green 1 vial 1 2 mL Table 4 Consumable Items Item Supplier and Catalog Number Quantity Spin Filters Costar Cat 8160 10 Ladder Tubes Genemate Cat C 3258 1 20 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 16 0 75 mL NC Table 5 DNA LabChips Item Catalog Number DNA Extended Range Chip for use with GX Cat 760517 Touch GXII Touch HT DNA Extended Range Chip for use with GX Cat CLS138948 Touch GXIl Touch 24 PN CLS140158 Rev C DNA High Sensitivity 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 products are used in accordance with the principles of good laboratory practice As al
3. 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 um filtered water Milli Q or equivalent water used for chip preparation e Replace the buffer used for sample and reagent preparation e Use a0 22 micron filter for all water and buffers used for chip sample and reagent preparation e Spin down sample plate to pellet any particulates Symptom Humps in several electropherograms which do not correspond to sample data 1250 1000 an o Fluorescence T o mhor 1 4 6 8 9 1 1 20 25 30 35 40 45 50 55 60 Time seconds Figure 18 Humps in several electropherograms PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Troubleshooting 24 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 expected 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 Normal migration time windows for the markers are e DNA High Sensitivity assay Lower Marker 21 25 seconds e DNA High Sensit
4. Ladder Tube 750 uL DNA sample buffer gt Buffer Tube a Ld LabChip GX Touch GXII Touch Figure 1 Standard Sample Workflow 1 Gently vortex DNA Ladder yellow cap for 10 seconds Briefly spin the ladder vial 2 Inthe provided 0 2 mL Ladder Tube add 12 uL of DNA Ladder to 108 uL of 1X DNA sample buffer solution Mix thoroughly by pipetting the solution up and down several times Ensure there are no air bubbles in the Ladder Tube 3 Insert the Ladder Tube into the ladder slot on the LabChip GX Touch GXIl Touch instrument PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 9 4 Pipette samples into a 384 well or 96 well plate Recommended sample volumes are 15 uL for a 384 well plate or 40 uL for a 96 well plate Seal and spin the plate at 1000 x g for 2 minutes prior to placing on the instrument 5 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 6 Insert the Buffer Tube into the buffer slot on the LabChip GX Touch GXIl Touch instrument Limited Sample Workflow 0 2X sample 384 well sample plate 2 uL sample 8 uL water only up to 96 wells can be tested 1X Ladder 12 uL DNA Ladder 108 uL DNA gt H 6 sample buffer Ladder Tube 150 uL DNA sample buffer 600 uL water gt A Buffer Tube TT ia r LabChip
5. Aligned Time sec Figure 15 DNA High sensitivity ladder electropherogram produced using the Limited Sample Workflow PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Troubleshooting 20 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 27 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 27 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 sample 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 Ifthe plate has been uncovered for some time sample evaporation might have occurred 5 Debris from the sample or
6. Close the chip door securely Touch the Wash button on the Home screen Figure 20 DNA High Sensitivity Assay User Guide LabChip Kit Essential Practices 30 Chip Status Type HT DNA High Sens Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 20 Wash screen After the completion of the wash cycle return the chip to the chip container ensuring the sipper is immersed 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 e Add Gel Dye solution to chip wells 3 7 8 and 10 using a Reverse Pipetting Technique as shown in Figure 19 Add 50 uL Low throughput or 75 uL High throughput DNA Marker green cap to chip well 4 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 e Close the chip door securely Touch the Run or Prime button on the Home screen PN CLS140158 Rev C DNA High Sensitivity Assay User Guide LabChip Kit Essential Practices 31 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 suc
7. 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 CLS140158 Rev C DNA High Sensitivity Assay User Guide Licenses and Rights of Use 35 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 the buyer the non transferable right to use the data for internal research and training purposes solely in combination with PerkinElmer Inc 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 limit
8. Marker Gel Dye Figure 4 Reagent placement Figure 5 Reagent placement for Low throughput up to 48 for High throughput up to samples 384 samples PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 12 6 Add DNA HiSens Marker green cap to chip well 4 as shown in Figure 4 Low throughput or Figure 5 High throughput 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 Make sure the rims of the chip wells are clean and dry IMPORTANT Ensure chip well 1 waste well is empty before placing the chip into the instrument Note Use the Low throughput protocol when running the LabChip GX Touch GXII Touch 24 instrument Inserting a Chip into the LabChip GX Touch GXIl Touch Instrument j Check that the sample plate Buffer Tube and Ladder Tube are placed on the instrument properly 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 Touch the Unload Chip button on the Home screen Chip Status Type HT DNA High Sens Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 2000 Samples Left Status Figure 6 Home screen PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 13 4 Insert the chi
9. diluted sample buffer solution in the Buffer Tube The sample plate should be tested soon after preparation to minimize evaporation Sipping samples more than once is not recommended Use this workflow if testing LabChip XT fractionated samples Quantitation given by the LabChip GX Touch software should be multiplied by the dilution factor i e your sample dilution ratio Preparing the Chip 1 Allow the chip to come to room temperature 2 Use a pipette tip attached to a vacuum line to thoroughly aspirate all fluid from the chip wells see Figure 3 For more details on how to set up a vacuum line see page 33 PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 11 Figure 3 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 Do not allow active wells to remain dry 4 If 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 or alcohol to clean the chip detection window as needed 5 Using a reverse pipetting technique add Gel Dye solution to chip wells 3 7 8 and 10 as shown in Figure 4 Low throughput 0 R75 or Figure 5 High throughput OOO Marker Gel Dye
10. GX Touch GXII Touch Figure 2 Limited Sample Workflow 1 Gently vortex DNA Ladder yellow cap for 10 seconds Briefly spin the ladder vial 2 Inthe provided 0 2 mL Ladder Tube add 12 uL of DNA Ladder yellow cap to 108 uL of diluted DNA sample buffer solution Ensure there are no air bubbles in the Ladder Tube Note Dilute 1X DNA sample solution at the same ratio as your samples with water 3 Insertthe Ladder Tube into the ladder slot on the LabChip GX Touch GXII Touch instrument PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 10 4 In a 384 well plate pipette 8 uL of water to an equivalent number of wells as samples being tested but to no more than 96 wells Add 2 uL of sample to each well and mix by pipetting up and down few times Avoid creating air bubbles Seal and spin the plate at 1000 x g for 2 minutes 5 Inthe provided Buffer Tube add 150 uL of 1X DNA sample buffer solution to 600 uL of water 6 Insert the Buffer Tube into the buffer slot on the LabChip GX Touch GXIl Touch instrument Notes This workflow requires only 2 uL of sample with a minimum initial concentration of 20 pg uL per fragment or 200 pg uL for smears Before testing the 2 uL of sample is diluted to 0 2X in water for a total volume of 10 uL To ensure a buffer match the sample buffer solution should be diluted in the same manner as your samples Prepare Ladder with the diluted sample buffer solution Use
11. N ond Fr ak ve Fr og SR 17 Chip Cartridge Cleaning mussresessrrreerssrrarernrrrrrerrrnrrrrrrr rr rr rrr rr r rr rn r RR RKA KRK RR RAK K Kr RR rr non 18 RE SUIS sisisi snatssngenenenieskentsnersesien snbsdskrnnsb pessen sanl nkin srs ekens rnsdskENabbe SenAFs RR Tis nER AGS 19 DNA High Sensitivity Ladder Result mossmsressessrrsrersrrrrrrrrrrrrrrrrrrrrrrrrn rr r rr enn rn rr rr nr ann 19 TEGUB ISSO OUI G ssasssereneansnan sus essensen eds kn nes denss RENARE GKS EE Homa SNR ER ARNNSE RANN SS eNeRT ee eee 20 Frequently Asked QUEeStiONS ssssssssssssssanssannnnrannnnsnann resan nns annen Ann Ran ARR nn RR Rn nerna 26 LabChip Kit Essential PraCtiCES sssssssssssssssssssssrnrensnsrrnrnnnnsnr nn nns RKA RR RR KAR RR RR NR 27 GENES AL ieir oeei eaka nE seed enceny ERE OE De ARNES NEDRE erfa RE E PE RETTE EE 27 Reagents anre enades tues a A aa A AA aA aaa aE ASE 28 CMD e E A E A A E E E 29 Sample S inei a a E A eee A en 32 Chip Well Aspiration Using a Vacuum sssssssssensnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn RR RR nn 33 Customer Technical Support s ssssssssssssrrsrssssssrrnnnnsnrnrnnnn ee ee heck eee 34 Licenses and Rights Of Use s ssssnsssssssssssssrrsssssrrssssrsnnrrnnnrnnn kk nn AREA KKR RAR ARR R KRA REAR NRK Rn Ar 35 PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Specifications 3 Specifications Assay Specifications Table 1 Assay Specifications Sizing Range 50 5000 bp Sizing Resolution
12. 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 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 Q or equivalent Add Marker Solution green cap to chip well 4 DNA High Sensitivity Assay User Guide Troubleshooting 22 e Reinsert the chip back into the instrument e Restart the run 2 Perform a marker channel unclogging procedure by repriming the chip See LabChip Kit Essential Practices on page 27 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 16 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 27 for instructions on how to reprime the chip PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Troubleshooting 23 Symptom Unexpected sharp peaks 600 Lower Upper marker marker Peak caused by particulates Fluorescence w o S 40 45 50 55 Aligned Time sec Figure 17 Unexpected sharp peak Possible causes
13. ed 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 DNA 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 Chip Status Type HT DNA High Sens Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 13 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 CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 18 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 CLS140158 Rev C a To reduce pressure leaks at the chip interface clean the O rings frequently Remove the O rings from the top pla
14. ed 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 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 CLS140158 Rev C DNA High Sensitivity Assay User Guide j gt PerkinE PerkinElmer Inc 68 Elm Street Hopkinton Massachusetts 01748 U S A TEL 508 435 9500 FAX 508 435 3439 http www perkinelmer com
15. hip Life 2000 Samples Left Buffer H hi Fol Assay Folder Change Assay Folder C Program Files x86 PerkinElmer LabChip GX Touch Assay Assay HT DNA High Sensitivity v Run Test Ladder after Prime Prime Figure 9 Chip priming screen 1 Touch the Run button see Figure 9 2 Select the appropriate assay type see Figure 8 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 10 Touch the green arrow button PN CLS140158 Rev C DNA High Sensitivity Assay User Guide 3 PN CLS140158 Rev C Preparation Procedures 15 Run Select Wells Setup Run Start Run Change Assay Fold Assay Folder C Program Files x86 PerkinElmer LabChip GX Touch Assay weet Select Assay Select Plate Type FSA tai rt Run Fil HT DNA High Sensitivity BioRad 96 HSP 96xx Sip 4 mm LSE AUS 123 45 6 7 8 9 101112 al Quadrants Status OO Column Wise Selection Sip Order Adv Settings A B C D E F G H 96 of 96 selected Figure 10 Selecting wells In the 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 11 Touch the green arrow button DNA High Sensitivity Assay User Guide Preparati
16. hout 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 Up to 96 samples in a 96 well or 384 well plate can be run with a single chip preparation Note The number of samples per chip prep is 96 A 384 well plate may be used to conserve sample volume but only 96 wells of the 384 can be tested PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Chip Well Aspiration Using a Vacuum 33 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 22 To avoid contamination use a new disposable pipette tip over the permanent tip for each chip aspirated Figure 23 Figure 22 A Permanent pipette tip attached to a house vacuum line B vacuum line with trap Figure 23 Replacing the disposable pipette tip PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Customer Technical Support 34 Customer Technical Support PerkinElmer
17. interface and clean if necessary PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Frequently Asked Questions 26 Frequently Asked Questions 1 PN CLS140158 Rev C Why is 120 uL of gel dye needed in Chip Well 10 for the DNA High Sensitivity assay High throughput workflow and not the other DNA assays As a result of the formulation of the DNA High Sensitivity assay additional ions are needed Why is 75 uL of Marker solution needed to run 96 samples using the DNA High Sensitivity assay High throughput workflow and only 50 uL of Marker solution for the other DNA assays For the DNA High Sensitivity assay more marker and sample are sipped compared to the other DNA assays If my DNA samples fall within the sizing range and linear concentration range of both the DNA 1K and the DNA High Sensitivity assays which assay should use The DNA High Sensitivity assay should be used since less sample is required to run this assay If the sample exceeds the assay concentration limits of the DNA High Sensitivity assay then it can be further diluted and re tested Why can only 96 samples be tested per High throughput chip preparation for the DNA High Sensitivity assay and up to 384 samples for the other DNA assays For the DNA High Sensitivity assay ion depletion occurs past 96 samples Additionally since more marker and sample are sipped compared to the other DNA assays the waste well in the chip fills up more quickl
18. ivity assay Upper Marker 54 64 seconds 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 within 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 27 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 PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Troubleshooting 25 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 27 for instructions on how to wash and reprime the chip 4 Check the O rings on the top surface of the chip
19. j gt Perkin DNA High Sensitivity 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 CLS140158 Rev C Contents Specifica lOH e ta beses ae ered na eee doo des ks Aer Rn aia 3 ASSAY SPCCIICATONS ecessesces cores cceeacaaeusscaseens dee Beacon cones datan bad a Ea 3 Sample Conditions resien VARAR Baer pe ER EA a E E e E AK RR 4 Kit Contents senenn eE aaa a E pa aa aaaeaii 5 Safety Warnings and PrecautionS sssssssnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn RR NAR 6 Preparation Procedures s ssssssssssasssssrsssssssnssnnn annan Han n kk nan KARNA RER ANAR ARR nnmnnn nnmnnn enne 7 Additional Items Required ovsesmssssssrsssssrrssrrssrrrorrrrosnnsrnrrrnn rr rn R RRD RR Ross R KKR KRK R RR KR DDR Krona 7 Preparing the Gel Dye Solution mlseesrererssrrrressrrrrreorrrrrrrrsnrrrrrrn rr rr r rr rr nr KRK rr RR RR rer on na 7 Preparing the DNA Samples DNA Ladder and the Buffer Tube 008 8 PEG ar ING UNAS aT o Me Pe rads eae sd E skador ns derek haa anda Aen hee ee 10 Inserting a Chip into the LabChip GX Touch GXIl Touch Instrument 12 Running the Assay missesseseesrrresrsrerrrrorrrnorsrsrsnrrrr AR Kro Arns se RR KAR ARK RAR RDR ARR RRR KRK KRK KKR RR KKR rna 14 Storing the Chp riean a ehe fee Ser Safe DARRE
20. l chemicals should be considered potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing such as 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 CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 7 Preparation Procedures Additional Items Required 18 megohm 0 22 um filtered water Milli Q or equivalent e 70 isopropanol solution in DI water Bio Rad Hard Shell 384 well Skirted PCR Plates Cat HSP 38XX recommended e 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 about 20 30 minutes before use Preparing the Gel Dye Solution Notes The Dye Solution contains DMSO and must be thawed completely before use The dye is light sensitive Do not expose the Dye solution or Gel Dye to light for any length of time Keep the prepared Gel Dye solution in the dark Do not exceed 9300 rcf when filtering the Gel Dye solution Exceeding 9300 rcf will change the properties of the gel One vial of DNA HiSens Gel Matrix red cap is good for 4 Low throughput chip preparations or 2 High throughput chip preparatio
21. n 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 PerkinEImer 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 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 High Sensitivity Assay User Guide LabChip Kit Essential Practices 28 Reverse Pipetting Technique Reagents PN CLS140158 Rev C Fv 4 STEP 1 STEP 2 STEP 3 STEP 4 Figure 19 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 aliquot
22. n 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 Sample Conditions 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 10 mM Tris and 1 mM EDTA for the DNA High Sensitivity assay Higher salt concentrations and different ions may alter performance and reduce assay sensitivity PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Specifications 5 Kit Contents Storage When not in use store chips and reagents refrigerated at 4 C Do not leave chips and reagents unrefrigerated overnight DNA High Sensitivity Reagent Kit Part Number CLS760672 Each kit contains enough reagents for 20 Low throughput LT or 10 High throughput HT chip preparations Up to 48 samples can be tested with a LT chip preparation Up to 96 samples can be tested with a HT chip preparation
23. ns Up to 48 samples can be tested with a LT chip preparation Up to 96 samples can be tested with a HT chip preparation 1 Vortex the thawed DNA Dye Concentrate blue cap for 10 15 seconds before use 2 Transfer 13 uL of DNA Dye Concentrate blue cap to 1 vial of DNA HiSens Gel Matrix red cap 3 Vortex the solution until it is well mixed and spin down for a few seconds Transfer the mixture into two spin filters approx 550 uL each Centrifuge at 9300 rcf for 10 minutes at room temperature Discard filters label and date the tubes N O Oo Ff Store in the dark at 4 C Use within 3 weeks PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 8 Preparing the DNA Samples DNA Ladder and the Buffer Tube Notes Total salt concentration must not exceed 10 mM Tris and 1 mM EDTA Higher salt concentrations and different ions may alter performance and reduce assay sensitivity 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 is the user s DNA buffer such as the PCR buffer etc Standard Sample Workflow Recommended sample 96 well sample plate volumes or 15 uL for a 384 well plate P 384 well sample plate 40 uL for a 96 well plate only up to 96 wells can be tested lt lt 1X Ladder 12 uL DNA Ladder O p 108 uL DNA sample buffer
24. on Procedures 16 LabChip GX I Touch HT Silas Run Select Wells Setup Run Start Run Operator F Read Plate Barcode Data Path C Program Files x86 PerkinElmer LabChip GX Touch Data rowse Default lv Copy to C Program Files x86 PerkinElmer LabChip GX Touch DataCopy Create Daily Sub Directory m Auto Export Data File Name Labchip_2014 06 12_07 20 57 gxd File Prefix Project name E Computer Name F Barcode Iv Date Advanced Settings ET sample Names File im Expected Peak File Ww Excluded Peak File Figure 11 Run setup screen 4 Touch Start to begin the run LabChip GX II Touch HT o x Run Select Wells Setup Run Start Run Chip Status Type HT DNA High Sens Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 2000 Samples Left Run Parameters Operator IM Assay HT DNA High Sensitivity 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 12 Starting a run PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Preparation Procedures 17 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 submerg
25. p into the LabChip GX Touch GXII Touch instrument Figure 7 and close the chip door securely Figure 7 Chip in the LabChip GX Touch GXIl Touch instrument 5 Touch the Load Plate button on the Home screen Figure 6 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 8 Touch the desired assay and then touch OK This chip supports multiple assays Select one HT DNA High Sens HT DNA 1K HT DNA 12K HT gDNA Figure 8 Assay Choice menu Notes If performing multiple runs in a day in between chip preparations the chip should be washed using the instrument and Chip Storage buffer as described in Storing the Chip on page 17 Be sure to periodically clean the O rings on the top plate of the chip interface on the LabChip GX Touch GXII 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 CLS140158 Rev C DNA High Sensitivity Assay User Guide Running the Assay Preparation Procedures 14 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 Chip Status Type HT DNA High Sens Chip Reagents N A Chip Expiration Jan 1 2020 C
26. s 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 High Sensitivity Assay User Guide Chips Repriming Chips LabChip Kit Essential Practices 29 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 CLS140158 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 Chip Storage Buffer to each active well 1 3 4 7 8 and 10 Place the chip in the LabChip GX Touch GXI Touch instrument
27. sample prep is clogging the sipper What to do 1 Add more sample to the well PN CLS140158 Rev C DNA High Sensitivity Assay User Guide Troubleshooting 21 Manually insert a larger volume pipette tip 100 uL 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 27 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 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 CLS140158 Rev C on instrument for extended period of time Add or replenish the marker solution in the chip using the following procedure Touch the
28. te of the chip interface on the LabChip GX Touch GXII instrument Soak O rings in water Milli Q 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 rings into the chip cartridge DNA High Sensitivity Assay User Guide Results 19 Results DNA High Sensitivity Ladder Result The electropherogram of a typical DNA High Sensitivity ladder using the Standard Sample Workflow is shown in Figure 14 Between the upper and lower markers peaks in order of increasing migration time correspond to ladder fragments of 50 100 150 200 300 400 500 700 1100 1900 2900 and 4900 bp 1 Ladder2 Ladder2 Upper 7200 Marker Lower Marker Fluorescence 20 25 30 35 50 55 60 40 45 Aligned Time sec Figure 14 DNA High sensitivity ladder electropherogram produced using the Standard Sample Workflow The electropherogram of a typical DNA High Sensitivity ladder using the Limited Sample Workflow is shown in Figure 15 2 Ladder Ladder 1600 Lower Marker 1400 Upper 1200 Marker 7 Fluorescence A m ao o Oo OO oOo Oo CJ Oo D no p o O 20 25 30 35 5 50 55 60 C 40 4
29. tion the sipper with a vacuum line as shown in Figure 21 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 21 Removing an air bubble or clog by suctioning the sipper with a vacuum line Other Considerations PN CLS140158 Rev C Chips should be stored refrigerated prior to first use Cover the active wells on the chip with Parafilm 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 High Sensitivity Assay User Guide Samples LabChip Kit Essential Practices 32 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 wit
30. y If using the DNA High Sensitivity assay to test samples fractionated by the LabChip XT what buffer do use to prepare the ladder and buffer for the Buffer Tube To test LabChip XT fractionated samples use the Limited Sample Workflow To prepare the ladder and buffer for the Buffer Tube use LabChip XT Running Buffer as the DNA Sample Buffer After removing the extracted collection from LabChip XT chip pipette out 300 uL of Running Buffer from source well DNA High Sensitivity Assay User Guide LabChip Kit Essential Practices 27 LabChip Kit Essential Practices General PN CLS140158 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 whe
Download Pdf Manuals
Related Search