Genomic DNA Assay User Guide
Contents
1. 18 Genomic DNA Software Analysis sse 18 Genomic DNA Ladder Result rm ne acne Pere Re ERE 18 Genomic DNA Res lt seenen tee port utes pua end Rs skid ere Dr ula 19 Troubleshooting issena rn sinn Rer daner ania uS anti cis ore dde KR Ne nin SR 20 LabChip Kit Essential PraCtiCeS s sssssssssssssssssssssrrrssnsrrnrnnnn rr r nn nns RKA RR RAKARE RR RNA 27 GEN STAN 2s E E suasaaites civaysccandew ade Es E E dees 27 Iai I e 9 M M 28 GHIPS eR I D I LIT 29 rera UO 32 Chip Well Aspiration Using a Vacuum eeeeeee nennen nennen nnn 33 Customer Technical SUDDOEL nire hr le eee 34 Licenses and Rights Of USB ria aue oes had dator Cs Ra V ORS ARR RAR RR RAR ARR USUS canes 35 PN CLS140166 Rev C Genomic DNA Assay User Guide Specifications 3 Specifications Assay Specifications Table 1 Assay Specifications Sizing Range 50 40 000 bp Sizing Accuracy 20 Up to 10 kb based on Sizing Precision 20 CV ladder Quantitation 0 2 5 ng uL For water as sample Range buffer Final concentration after dilution Sensitivity 0 1 ng uL S N 3 intact Human Control gDNA Quantitation 3096 Based on PicoGreen Accuracy quantitation of Human Control gDNA Quantitation 2096 CV Based on Human Control Precision gDNA Sample Volume 10 uL minimum Requires2. oste Start Run Advanced Settings m Sample Names File m Expected Peak File Excluded Peak File Figure 11 Run setup screen 4 Touch Start to begin the run PN CLS140166 Rev C LabChip GX II Touch HT Run Chip Status Type Chip Reagents Select Wells Setup Run Start Run HT gDNA N A Chip Expiration Jan 1 2020 Chip Life Run Parameters Operator Assay Data Path Plate Name Barcode Plate Cycles Auto Export Sample Saver Random Selection No 500 Samples Left Irina HT gDNA C Program Files x86 PerkinElmer LabChip GX Touch Data BioRad 384 HSP 38xx Ver 2 Sip 2mm N A 1 No No Figure 12 Starting a run Genomic DNA Assay User Guide Preparation Procedures 16 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 St
3. is much longer than expected Delayed Migration Example A6 A6 Type 20 1 Lower 1 Marker BT Genomic DNA 50 i Rae MUT I fi 1 51 T T T T T att 0 0 0 1 03 0 7 11 19 29 49 7 0 10 0 40 0 T 05 Size kb Figure 22 Electropherogram of genomic DNA migration that is too slow Possible causes 1 Particulates from the samples may be clogging the separation channel 2 Excess dye within the separation channel 3 Gel Dye was not primed properly into the chip What to do 1 Minimize the loading of particulates in the sample by spinning down the plate e g 3000 rcf for 5 minutes before testing and or selecting a plate type with a higher sip height in the Start Run dialog box before starting a new run The debris maybe flushed out of the chip by washing and repriming the chip 2 Prepare a fresh Gel Dye solution Wash the chip and then reprime with the new Gel Dye mixture 3 Check the O rings on the top surface of the chip interface and clean if necessary then reprime the chip PN CLS140166 Rev C Genomic DNA Assay User Guide LabChip Kit Essential Practices 27 LabChip Kit Essential Practices General PN CLS140166 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 well
4. or 2 Large batch chip preparations Up to 24 samples can be tested with a Small batch chip preparation Up to 48 samples can be tested with a Large batch chip preparation 1 PN CLS140166 Rev C Vortex the thawed Genomic DNA Dye Concentrate blue cap 9 for 10 seconds before use Transfer 13 75 uL of Genomic DNA Dye Concentrate blue cap to 1 vial of Genomic DNA Gel Matrix red cap Vortex the solution until it is well mixed and spin down for a few seconds Transfer the mixture into two spin filters 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 Genomic DNA Assay User Guide Preparation Procedures 8 Preparing the DNA Samples DNA Ladder and the Buffer Tube Sample Workflow 384 well Sample Plate Samples should be diluted in water Maximum samples per run 48 A 96 well plate can be used but it requires a minimum of 20 uL and a low sip height See Additional Items Required section for plate type compatibility 1X Ladder 12 uL DNA Ladder 108 pL water Note Do not vortex ladder Fadder Tube solution Vortexing may degrade large DNA ladder fragments 750 uL water gt Ln Buffer Tube a LabChip GX Touch GXII Touch Figure 1 Sample Workflow 1 Inthe provided 0 2 mL Ladder Tube add 12 uL of Genomic DNA Ladder yellow cap to 1
5. 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 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 CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 13 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 X Change Assay Folder R Chip Status Assay Folder 9 y Type HT gDNA C Program Files x86 PerkinElmer LabChip GX Touch Assay Chip Reagents N A Assay Chip Expiration Jan 1 2020 HT gDNA Chip Life 500 Samples Left Run Test Ladder after Prime Status Buffer Views p 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
6. to air dry Reinsert the O rings into the chip cartridge Genomic DNA Assay User Guide Results 18 Results Genomic DNA Software Analysis Data are analyzed by aligning the sample data and normalizing the sample area using the Lower Markers in the samples and in the two ladders that bracket every 12 samples The size of a sample is determined by comparing the migration times of peaks within the sample to those of the fragments of known size in the bracketing ladders The concentration of a sample is calculated using a Total gDNA smear starting at 0 18 kb and extending to 300 kb see Figure 14 These smear limits can be adjusted by the user A calibration curve generated using the known size and concentrations of ladder peaks are applied to the normalized area of this Total gDNA smear to determine the concentration of the sample This Total gDNA Concentration is reported in the Well Table entry of each sample The Genomic DNA assay also reports a Genomic DNA Quality Score GQS in the Well Table entry of each sample This score represents the degree of degradation of a sample with 5 corresponding to intact gDNA and 0 corresponding to highly degraded gDNA and is calculated from the size distribution of a sample Total gDNA Smear B5 B5 so TY Fluorescence T T T T T tao 0 0 0 1 03 05 07 11 19 29 49 7 010 040 0 Size kb Figure 14 Total gDNA smear in orange Genomic DNA Ladder Result The el
7. 08 uL water Milli Q or equivalent 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 Note Do not vortex the ladder solution Vortexing may degrade large DNA ladder fragments 2 Insert the Ladder Tube into the ladder slot on the LabChip GX Touch GXIl Touch instrument 3 Prepare samples in 384 well plates Add 2 uL of sample to 18 uL of water Milli Q9 or equivalent If sample volume is limited 1 uL of sample can be added to 9 uL of water PN CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 9 Notes Due to evaporation samples prepared with 1 uL of sample and 9 uL of water should only be tested in Small batch runs 24 samples Samples at concentrations from 0 2 to 5 ng uL should be run undiluted Do not exceed 5 ng uL final concentration in the well as this can clog the chip channels We recommend testing intact DNA at 2 5 ng uL 4 Add 750 uL of water Milli Q or equivalent to the 0 75 mL Buffer Tube provided with the reagent kit Ensure there are no air bubbles in the Buffer Tube 5 Insert the Buffer Tube into the buffer slot on the LabChip GX Touch GXIl Touch instrument Buffer Tube Ladder Tube L Sample CJ OR OK OR JK JE JK JK JK JK RJ Well A1 v 00000000 0 0 VN VU NUUS SY AAA III IF YY IF Ladder Tube Buffer Tube Figure 2 Locations of the Buffer Tube and Ladder Tube
8. 25 Symptom Humps in several electropherograms which do not correspond to sample data Fluorescence 1250 1000 750 500 250 y 0 4 pisci deer rr Pe T C L 1 20 25 30 35 40 45 50 55 60 Time seconds Figure 21 Humps in several electropherograms 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 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 Possible causes 1 Incorrect Gel Dye concentration What to do 1 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 Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture 2 If fast migration is observed repeatedly on a new chip contact PN CLS140166 Rev C Technical Support Genomic DNA Assay User Guide Troubleshooting 26 Symptom Peaks migrating much slower than expected An electropherogram of a genomic DNA sample is shown in Figure 22 where the migration time
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10. Ba Perkin Genomic DNA 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 CLS140166 Rev C Contents Specifica lOH 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 ins noie erc tara eti nde lada xtd ea ed ce s Pa ud E mannen ne 7 Additional Items Required oro iro pi date aede Cedere de trea beni ate DR RK Nona 7 Preparing the Gel Dye Solution mlseeseererssrrrressrrrrrerorrrererrrnrrrrrrn rr rr r rr narr KRK rr RAR nr nn na 7 Preparing the DNA Samples DNA Ladder and the Buffer Tube 8 Preparing NewS MD fon eres cate AP Ulta ol iL SE b CE E 9 Inserting a Chip into the LabChip GX Touch GXII Touch Instrument 11 Running the Assay osseeerrereesrerereerenrrnrrerrrrnrrnrrnrrrnr rens nr rr ene hem rem nemen nn nnne 13 Storing The I Ta eerte Pe PEE SD tab n Pues RO eet oer I Dots RN Sr 16 Chip Cartridge Cleaning 3 sse Ove eerta tix ehe te ee RR KK Kr Ren n on 17 RE SUIUS ge e
11. a 384 well plate Required 20 uL A 96 well plate can be recommended used but it requires a minimum of 20 uL and a low sip height Samples per 48 or 24 Two workflows one for Chip Prep 24 samples one for 48 samples Analysis Time 48 samples in 2 5 Walk away time hrs Samples per 480 Chip Reagent Kit Chip Reagent Kit 3 9 months Stability 1 Human Control gDNA from intestine was purchased from BioChain Hayward CA PN CLS140166 Rev C Genomic DNA Assay User Guide Specifications 4 Sample Conditions Table 2 Sample Conditions Additives PerkinElmer recommends that BSA and detergents exceeding 0 05 mg mL and 0 0196 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 All sample plates should be spun down prior to analysis All buffers should be filtered with a 0 22 um cellulose acetate filter Salt Concentration Total salt concentration must not exceed 10 mM Tris Higher salt concentrations and different ions may alter performance and reduce assay sensitivity Kit Contents Storage When not in use store chips and reagents refrigerated at 4 C Do not leave chips and reagents unrefrigerated overnight Kit contains enough reagents for 20 Small batch or 10 Large batch chip preparations Up to 24 samples can be tested with a Smal
12. adjusted by clicking and dragging 7 35 j Baseline Adjustment A6 A6 Default Baseline st GQS 3 3 Total gDNA Conc 0 89 ng uL Fluorescence st t SSI 0 0 0 1 0 3 0 5 0 7 11 19 29 4970 100 40 0 Size kb Baseline Adjustment A6 A6 Adjusted Baseline 25 e GQS 3 0 Concentration 1 07 ng uL Fluorescence 54 L L3 0 0 0 1 03 05 07 11 19 29 4970 100 400 Size kb Figure 17 Electropherograms of sample with a poor baseline fit and an adjusted baseline Symptom No ladder or sample peaks but marker peaks detected Note The lower marker peak height will most likely be greater than normal height PN CLS140166 Rev C Genomic DNA Assay User Guide Troubleshooting 21 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
13. atus Type HT gDNA 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 CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 17 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 CLS140166 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
14. 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 sample prep is clogging the sipper What to do 1 Add more sample to the well 2 Manually insert a larger volume pipette tip 7100 uL into the sample well and dislodge the bubble Rerun these sample wells 3 Check the plate definitions 4 Check the sample wells especially around the edge of the plate where evaporation is fastest and make a fresh plate if volumes are low PN CLS140166 Rev C Genomic DNA Assay User Guide Troubleshooting 22 5 f 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
15. due to a marker channel clog What to do 1 This may be due to not filling marker well or chip remaining idle on instrument for extended period of time Add or replenish the marker solution in the chip using the following procedure 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 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 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 27 for instructions on how to reprime the chip PN CLS140166 Rev C Genomic DNA Assay User Guide Troubleshooting 23 Symptom Ladder traces show up in the lanes following the ladders delayed sip b 4 Algoed Time sec Figure 18 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 Symptom Unexpected sharp peaks Electropherograms of genomic DNA are sho
16. ectropherogram of a typical Genomic DNA ladder is shown in Figure 15 Following the Lower Marker are ladder fragments of 100 300 500 700 1100 1900 2900 4900 7000 10000 and 40000 bp PN CLS140166 Rev C Genomic DNA Assay User Guide Results 19 gDNA Ladder Ladder1 Ladder1 390 Lower J Marker tu LA t t t 0 0 0 1 03 y 19 29 49 70 100 400 ee kb Figure 15 Genomic DNA Ladder Genomic DNA Result An electropherogram and gel view of intact and degraded genomic DNA from BioChain is shown in Figure 16 ue DNA was degraded by incubating with Fragmentase for 10 or 20 minutes Genomic DNA treated for 10 minutes was partially degraded GQS 2 5 while genomic DNA treated for 20 minutes was highly degraded GQS 0 3 Intact gDNA GQS 4 5 t g 10 min Degraded gDNA E L i S 20 min Degraded gDNA I i GQS 2 5 i GQS 0 CC MO ee Figure 16 Intact and degraded genomic DNA from BioChain PN CLS140166 Rev C Genomic DNA 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 Unexpected concentration and or GQS Possible causes and what to do 1 If an unexpected concentration and or GQS value is obtained check the baseline of gDNA smear for a proper fit Baselines can be manually
17. hat 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 all 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 CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 7 Preparation Procedures Additional Items Required 18 megohm 0 22 m 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 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 One vial of Genomic DNA Gel Matrix red cap is good for 4 Small batch
18. in the GX Touch GXII Touch instrument 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 CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 10 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 Milli Q or equivalent 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 Milli Q or equivalent 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 For Small batch add 50 uL per well as shown in Figure 4 For Large batch add 75 uL in wells 3 7 and 8 and 120 pL in well and 10 as shown in Figure 5 0 R 009 4 OO XO 7 Marker Gel Dye Marker D Gel Dye Figure 4 Reagent placement Figure 5 Reagent placement for Small batch up to 24 for Large batch up to 48 samples samples PN CLS140166 Rev C Genomic DNA Assay User Guide Prepara
19. l batch chip preparation Up to 48 samples can be tested with a Large batch chip preparation Table 3 Genomic DNA Reagent Kit Contents PN CLS760685 Reagent Vial Quantity DNA Dye Concentrate Blue Q 1 vial 0 09 mL Chip Storage Buffer White 9vials 1 8 mL each Genomic DNA Gel Red Q 5vials 1 1 mL each Matrix 10X Genomic DNA Yellow 1 vial 0 26 mL Ladder Genomic DNA Marker Green Q 1 vial 1 5 mL PN CLS140166 Rev C Genomic DNA Assay User Guide Spec Table 4 Consumable Items ifications 5 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 20 0 75 mL NC Table 5 DNA Extended Range LabChip Item Catalog with GX Touch GXII Touch HT Number DNA Extended Range Chip gDNA for use Cat 760517 DNA Extended Range Chip gDNA for use Cat C with GX Touch GXII Touch 24 LS138948 PN CLS140166 Rev C Genomic DNA 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 t
20. new disposable pipette tip over the permanent tip for each chip aspirated Figure 27 Figure 26 A Permanent pipette tip attached to a house vacuum line B vacuum line with trap Figure 27 Replacing the disposable pipette tip PN CLS140166 Rev C Genomic DNA Assay User Guide Customer Technical Support 34 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 CLS140166 Rev C Genomic DNA 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 diag
21. ng and Repriming Chips PN CLS140166 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 24 Genomic DNA Assay User Guide LabChip Kit Essential Practices 30 Chip Status Type HT gDNA Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 24 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 Add Gel Dye solution to chip wells 3 7 8 and 10 using a Reverse Pipetting Technique as shown in Figure 23 Add 60 uL Small batch or 120 uL Large batch DNA Marker green cap to chi
22. nostic 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 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 CLS140166 Rev C Genomic DNA Assay User Guide j gt Perkin PerkinElmer Inc 68 Elm Street Hopkinton Massachusetts 0174
23. onment 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 Genomic DNA Assay User Guide Samples PN CLS140166 Rev C 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 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 Up to 96 samples in a 96 well or 384 well plate can be run with a single chip preparation Genomic DNA 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 26 To avoid contamination use a
24. p 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 Close the chip door securely Touch the Run or Prime button on the Home screen PN CLS140166 Rev C Genomic DNA 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 suction the sipper with a vacuum line as shown in Figure 25 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 25 Removing an air bubble or clog by suctioning the sipper with a vacuum line Other Considerations PN CLS140166 Rev C Chips should be stored refrigerated 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 envir
25. s 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 Q9 or equivalent Using the Reverse Pipetting Technique described next will help avoid introducing bubbles into the chip when pipetting the gel PerkinElmer Inc 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 Genomic DNA Assa
26. tion Procedures 11 6 Add 60 uL Genomic DNA Marker green cap to chip well 4 for Small batch Figure 4 or 120 uL for Large batch Figure 5 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 placed on the instrument properly 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 gDNA Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 500 Samples Left Figure 6 Home screen 4 Insert the chip into the LabChip GX Touch GXII Touch instrument Figure 7 and close the chip door securely PN CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 12 Figure 7 Chip in the LabChip GX Touch GXII 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
27. well is sampled under Adv Settings Figure 10 Touch the green arrow button PN CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 14 Run Select Wells Setup Run Start Run Change Assay Fold Assay Folder CAProgram Files x86 PerkinElmer LabChip GX Touch Assay Send Select Assay Select Plate Type 2X2 X 22 I rt Run Fil Iur gDNA w BioRad 384 HSP 38xx Ver 2 Sip 2mm A CI iade Full Quadrants Size sosseesssseecce ccc DOOS uocem DE SOO899000000000000000 7 column wise es gem 33232838238 Sees S t 9C OCC J Adv Settings iun 24 of 48 selected Figure 10 Selecting wells 3 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 11 Touch the green arrow button PN CLS140166 Rev C Genomic DNA Assay User Guide Preparation Procedures 15 LabChip GX II Touch HT Run Operator Data Path Iv Copy to Iv Create Daily Sub Directory Data File Name File Prefix EJ Computer Name m Barcode Select Wells Setup Run C Program Files x86 PerkinElmer LabChip GX Touch Data rowse Default C Program Files x86 PerkinElmer LabChip GX Touch DataCopy E Auto Export Labchip_2014 06 13_04 36 06 gxd Project name V
28. wn in Figure 19 and Figure 20 with unexpected sharp peaks caused by particulates like dust or aggregates of large DNA that can form during sample loading and or migration in the chip PN CLS140166 Rev C Genomic DNA Assay User Guide Troubleshooting 24 7 dn i00 IS Peak caused by SH particulates or 3 us DNA aggregates w 1 Lower 250 4 Marker j 200 4 j 150 I 100 i 50 i r4 ka JA 1 e d WS od P A ES TE sd dans d VJ RD ERR us ua i 07 1 1 i 29 49 0 40 0 Size kb Figure 19 Electropherogram with an unexpected peak migrating after a genomic DNA sample om Je AL Peak caused by J L GwWar particulates or se Marker DNA aggregates i 300 PoE m E AN f 1 1 1 1 pop LE ALTAE t t t t t ot 0 0 0 1 03 0 5 11 19 29 49 7 0 10 0 40 0 07 Size kb Figure 20 Electropherogram with an unexpected peak that overlaps with the migration of a genomic DNA sample Possible causes 1 Dust or other particulates introduced through sample or reagents 2 Aggregates of large genomic DNA What to do 1 Replace the buffer used for sample and reagent preparation Use a 0 22 micron filter for all water and buffers used for chip sample and reagent preparation 2 Retest the sample Dilute sample further if possible to reduce aggregation PN CLS140166 Rev C Genomic DNA Assay User Guide Troubleshooting
29. y User Guide LabChip Kit Essential Practices 28 Reverse Pipetting Technique Reagents PN CLS140166 Rev C STEP 1 STEP 2 STEP 3 STEP 4 Figure 23 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 Genomic DNA 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 Washi
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