LabChip XT DNA Kit, Assay User Guide
Contents
1. LabChip XT DNA Kit Assay User Guide Collect Fractionated Material For multiple extractions During the pause after each collection open the LabChip XT instrument lid and pipette recovered DNA sample from each collection well into a clean tube for downstream processing If necessary wash well with 201 buffer and add 20yl buffer for next collection Close lid and click Resume For single extractions After run has completed open the LabChip XT instrument lid and pipette recovered DNA sample 8 from each MERIAL collection well into a clean tube for downstream processing Analyzing Data Refer to the LabChip XT User Manual for Data Analysis Caliper Life Sciences Page 9 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Calipe Y LabChip XT DNA Kit Assay User Guide Typical Results Example 1 Ladder and 3 channels with DNA of varying mass input and size selection eXtract and Stop mode XT 750 1 Channel 1 Caliper sizing ladder 2 Channel 2 250ng input select by size range 600bp 5 blue 3 Channel 3 100ng input select by size range 300bp 5 red 4 Channel 4 1000ng input select by size range 100bp 5 brown 2011 06 16 Exp1_Alameda_2011 06 16_05 46 29 C18 1863 1 2 C19 1863 1 3 Fluorescence 20 30 5070100 200300 500700000 Size BF Figure 1A LabChip XT Figure 1B LabChip GX Electropherogra2. 5007001000 pO E Figure 5A LabChip XT Electropherogram Figure 5B LabChip GX Electropherogram of collected material Example 6 Extraction of miRNA library Skip Extract and eXtract and Pause modes XT300 1 Channel 1 Caliper sizing ladder 2 Channel 2 100ng miRNA library input Step 1 Skip Extraction Peak Max Threshold at 25RFU min Width at 25bp grey Step 2 Extract and Pause Peak start Threshold at 5ORFU min Width at 5 red 1 IKT DNA 300 Ladder Sizing Ladder 100 190 502 300 MIRNA XT extractions A 7 XT Fractionated Libr 400 E Time thin 50 XT DNA 300 CH3 miRNA Lib 100ng 148 x Fluorescence m e o m 13870 g 791112 195 E 243g 3 5710 2030 507000 20000500 ze BP Time thin 50 Figure 6A LabChip XT Electropherogram Figure 6B GX electropherogram of collected material Caliper Life Sciences Page 12 of 24 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Troubleshooting e Chip fails connectivity test e Once a chip has failed a connectivity test record which channel has failed Remove the chip from the instrument o Check that the outlets leading from the chip to the buffer reservoirs are not blocked by an air bubble Air bubbles may be dislodged by gently tapping the chip near the location of the air bubb
3. conis dis eeu gas ausu UeE E DeUuiUsUswES SUN SOUTEg a E Sac cai Ce P Is EE Orbe SE e 8 STARTING THE EABCHIP E 8 nu PB ZAIN Gy asses een 9 Re eS mee 10 CHIP WELL ASPIRATION USING A VACUUM OR MANUAL PIPETTE 22 OPENING CHIP CONTAINER 23 OPENING CHIP CONTAINER WITH OPTIONAL LID 23 REORDERING INFORMATION 0 02c0ccccscesceensceenscncnsneceenscnensceenscnensnensnscnensenensenensenensenensnscnensenensensnsenensnsenensenenses 24 TECHNICAL SUPPORT uu ccceccececcececcecncescnceecncescecescecnsenenseeenseseuseseuceseaeesensnseesaseseaseseaseseasesensusenenseseaeeseueesenenseaeesenenss 24 Caliper Life Sciences Page 1 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Gui
4. gt Cali er LabChip XT DNA Kit Assay User Guide Table of Contents INTRODUCTION mo e 2 APPLICATIONS ccceccececcececcececcececucecuucecuucecueueuucuececuunecuunecsenecacausucuunecsunecseausseuececunecsunecsuaecsuaesaeaesecaunecsuaesseaeseteeeeraees 2 FEATURES ccccceccececcececcececcecucuccucuccecuccecuenecueaecseneuueuuecueuecsusecuenucueaeueueuecuunecuunecsuaesseaecseauseeeuecsesessuaeseeauseeaeeareeseraees 3 SPECIFICATIONS cceccecececccccececcccccececueeuececscueuuuececaeuuuenecsuuuaunenecseuunenensuaunenenecseauaunenenecsenenecsuauaunesecseuuaunenenenscauaunenenes 3 PTT ONT NGS M 4 ADDITIONAL ITEMS REQUIRED 0cccceccececcececcececcececuecucccecuececueaecuececseueuacausecsuuecuuaecsenecseeuecenecsunecsenecseausececsueeuseceeaees 4 SAFETY WARNING amp PRECAUTIONS ccccceccececcececcececcecucuececuccecuececsuaecaeaucucauuecseuecsenessueuecuuuecsesecsuaessuausseesucsunecsuaecass 5 SAMPLE PREPARATION PROCEDURE cccccecesceceececescecescecnccscnscecuceseuceseucnscaeesenenseeeuseseuseseneeseueeseneusenensesensesenens 6 CREATE LABGHIP HUN 6 PREPARE XI DNA CHIP
5. Caliper Life Sciences Page 21 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch m Caliper WV LabChip XT Kit Assay User Guide Chip Well Aspiration Using a Vacuum or Manual Pipette Removal of excess gel or buffer from the sample and collection wells can be accomplished by manual pipetting or by attaching a pipette tip to a house vacuum line or vacuum pump with trap Figures 8A Attach a pipette tip to the vacuum line and carefully aspirate excess gel Caliper recommends using a pipette tip with an opening of approximately 1 mm and regulating the vacuum to no more than 20 in Hg to prevent too high a flow rate and possible dislodging of gel in the channel Figure 8B During vacuuming avoid making a tight seal with the pipette tip at the bottom of the sample and collection wells as this could result in the removal of gel from the separation channel Figure 8B Attached pipette tip for vacuuming Figure 8A Vacuum setup of wells To prevent contamination use a new pipette tip over the permanent tip for each chip aspirated or a new pipet tip with each manual pipette Ensure the well bottoms are not blocked by gel or buffer before proceeding Figure 9 f T CIO Figure 9A Sample well after comb removal gel present Figure 9B Sample well after vacuuming void of all gel Caliper Life Sciences Page 22 of 24 PN 450713 Rev 08
6. Assay User Guide Reordering Information Product Part Number LabChip XT DNA 750 Assay Kit 760541 LabChip XT DNA 300 Assay Kit 760601 Technical Support Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 1668 Phone 1 877 LABCHIP 522 2447 Fax 1 508 435 3439 For additional assay and instrument troubleshooting refer to the LabChip HT Software Help file Call Caliper Technical Support at 1 877 LABCHIP 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 XT User Guide Caliper the Caliper logo LabChip and the LabChip logo are registered trademarks of Caliper Life Sciences This product is provided under an agreement between Life Technologies Corporation and Caliper Life Sciences Inc and the manufacture use sale or import of this product is subject to one or more US patents and corresponding non US equivalents owned by or licensed to Life Technologies Corporation and its affiliates The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product onl
7. the XT XTe Instrument Manual for operations modes available for your instrument a Disabled The channel will not be used in the run Any channel that has already been used is automatically set to Disabled and cannot be reused b Ladder The channel contains a ladder that is used to calculate the sizes in the samples in the remaining channels during the run c eXtract and Stop The sample in the channel will run until the extraction is complete After the extraction no additional sample will move through the channel d eXtract and Continue The sample in the channel will run until the end of the chip range The size range specified in the Extraction Region will be collected in the collection well e eXcLuDe Region The sample in the channel will be collected in the waste well on the chip except for the selected region which moves into the collection well on the chip The desired output is extracted from the waste well f Separation The sample in the channel will run until the end of the chip range No collection will occur g eXtract and Pause Used for multiple extractions the sample in the channel will be collected in the collection well and the run will pause for collection before resuming the run h Skip Extraction Skips over a known region in the sample without extracting any sample to the Collection well The software automatically moves to the next step in a multistep extraction immediately after the skipped region is co
8. DNA Kit Assay User Guide Sample Preparation Procedure The sample can be prepared up to 1 hour in advance of chip run either before or after the creation of the run file Caution 6x Sample Buffer is light sensitive Care should be taken to reduce light exposure of prepared sample 1 Allow Reagent Kit to equilibrate to room temperature before use approximately 20 to 30 minutes 2 Ladder preparation a Dilute 2uL of ladder O with 8uL of EB or TE Buffer Use the same diluent as the sample buffer b Add 2uL of 6X Sample Buffer C Vortex sample to mix Spin down 3 DNA Sample preparation a b C If necessary adjust volume of DNA sample 10uL by diluting with EB or TE Mix 10uL of DNA sample with 2uL of 6X Sample buffer Q9 Vortex sample to mix Spin down Create LabChip Run File This step can be prepared in advance of sample preparation and chip preparation The creation of the run file is not time sensitive 1 Launch LabChip XT XTe software On the LabChip XT XTe Main Window select Tools Run File Editor The Run File Editor window opens 2 Select the Method XT DNA 750 or XT DNA 300 to edit from the Fractionation Method drop down list Each Method is restricted to a type of chip ie DNA 300 or 750 3 Under the Setup Tab type the desired name for each channel in the Sample Name box 4 For each channel select the desired operation for the channel using the pull down menu Refer to
9. test under the Validation menu If a channel s gain is not adequate or background is too high contact Caliper Technical Support 35 100 200 300 400 600 1000 10380 e Optical detection was saturated during run Saturation may occur if more DNA is loaded than supported by the assay specifications Run an optics diagnostic test using the test chip under the Validation menu If any diagnostic failures are reported contact Caliper Technical Support e Collection well was empty after run Ensure that enough Collection Buffer was added to the collection well to completely fill the bottom of the well before the run has started The failure to add collection buffer however should result in a failed connectivity test Chip may have been left on instrument too long and sample might have dried over time It is important to recover the sample immediately after a run is completed to avoid loss of sample Laboratory temperature may be too high and sample is evaporating over the course of the run Incorrect reagent was used in the collection well Ensure that the collection buffer red vial was used e Collection well has more buffer than was added at the beginning of the run If the sieving material in the channel entering or exiting the collection well is disrupted buffer can continuously flow into the collection well See step 6 in the section Prepare XT DNA Chip above If buffer is flowing into the collection well the channel is considered to
10. 3 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 h Caliper LabChip XT DNA Kit Assay User Guide Prepare XT DNA Chip Note The dye contains DMSO and must be thawed completely before use Gently vortex and centrifuge all kit reagents before use jm 2 3 8 Remove Chip from foil bag and peel back top seal from chip Gently remove Sample Well and Collection Well combs If gel or buffer is present in either the sample or collection wells it should be manually removed before sample loading See Chip Well Aspiration Using a Vacuum or Manual Pipette at end of User Guide for illustrations SOURCE WASTE If a well fills with buffer after vacuuming do not use that SAMPLE wel S COLLECTION WELL channel If necessary up to 60 uL of buffer for rinsing and or collection can be taken from either the Source Reservoir or the Waste reservoir before the run and addition of dye Vortex the Dye and spin it down Pipette 15uL of the XT DNA Dye to the bottom of each of the four Waste Reservoirs Gently rock the chip to mix the dye and buffer Visually check to see that the dye has been evenly distributed throughout the reservoir Ensure the top surface of the chip is dry ADD DYE Steps 9 11 are time sensitive steps The run should be started within 5 minutes to prevent diffusion of loaded DNA sample and stacking buffer 9 10 Add 20uL of the Stacking Buffer to rectang
11. 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch m Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Opening Chip Container Manually To manually open the chip container first lift tab up until it is in a vertical position Do not rock the tab back and forth Next position the can so that it is held securely in place with the smooth side of the can against your body The tab should be positioned at 3 o clock Pull the lid back until it is detached Carefully reach in to remove the chips Opening Chip Container with Optional Lid Opener A lid opener is an optional accessory item P N 133524 that can be used to assist in opening of the XT chip containers To use the lid opener use the following steps Step 1 Open handles Attach lid opener parallel to top of container aligning the two arrows Be sure the cutting mechanism fits snugly against the side of the can Close handles Step 2 Turn knob clockwise After a full circle around the edge of container you will feel the resistance drop Step 3 To release turn knob slightly counter clockwise and open handles Step 4 Use the Lid Gripper small metal pliers on right side of can opener to easily lift lid from container Carefully reach in to remove the chips Caliper Life Sciences Page 23 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit
12. 748 1668 1 877 LABCHIP 1 877 522 2447
13. DNA Kit Assay User Guide Frequently Asked Questions Kit Specification What is in the Ladder The XT DNA Ladders are a mixture of individually chromatography purified double stranded DNA fragments The ladder is recommended for sizing and approximate quantification of a wide range of double stranded DNA The ladder is supplied in Storage Buffer TE buffer which is made up of 10 mM Tris HCl pH 7 6 and 1 mM EDTA Ladder data is used to convert the migration time of each sample peak into a size What is in the 6xSample Buffer The 6x Sample Buffer contains the lower marker first peak in the ladder DMSO and glycerol The lower marker is not a nucleic acid and will not interfere with sequencing Once the 6x Sample Buffer has been mixed with the sample of interest glycerol increases the density of the DNA sample and ensures that it will load below the stacking buffer It is imperative that the stacking buffer is not mixed with the DNA sample below to ensure proper stacking of the DNA What is in the collection buffer and is it compatible with sequencing The collection buffer is formulated with a Tris acetate based buffer containing a small amount of EDTA that is mixed with a polymer component which increases extraction efficiency It is slightly basic in pH 8 3 and is compatible with sequencing and downstream analysis What is the maximum amount of DNA that can be loaded in each channel The maximum amount of DNA that can be loa
14. Sciences abChip XT DNA Kit Assay User Guide Assay Preparation 1 How long can DNA remain in sample well after being loaded Caliper recommends that a user run the sample within 5 minutes of sample loading 2 How long can DNA remain in the collection well after being run Caliper recommends that a user collect the sample within 20 minutes of a run ending 3 Canluse my own buffer in the collection well Caliper does not recommend using alternate chemistries with XT reagents Standard reagents have been carefully optimized for best performance 4 Canluse my own dye Caliper does not recommend using alternate dye chemistries with XT chips and reagents 5 Canchips or individual channels be reused Chips and individual channels cannot be re used Once a channel has been used Caliper cannot guarantee that the chip will run properly or that there will not be any cross contamination The software automatically disables any channel that has previously been used to prevent accidental reuse 6 How dol maximize recovery of my DNA The best way to maximize DNA recovery is to stay within kit specifications do not over load channel with excess DNA practice proper sample preparation chip preparation DNA loading techniques homogeneous mixing of dye and buffer and use low salt DNA diluent for maximum stacking For maximum run speed and collection efficiency choose the extract and stop operation or use extract and pause when collecting multip
15. and collection wells before starting the test You should not run this test with the sample loaded as the sample will be pushed into the channel during the test After running the test you should carefully rinse the sample well and remove all liquid before proceeding with the normal chip preparation 11 What is the shelf life of the chips Chips are currently marked with a shelf life of 6 months from the date of manufacture Customers are guaranteed a shelf life of 4 months from the shipping date 12 Can store the chips in the refrigerator to extend shelf life of chips No chips should be stored at room temperature 18 28 C 13 I load more than 10uL of sample but stay under 1ug per channel To achieve the stated kit specifications 10 uL of sample is recommended However 15uL of sample per channel may be used with only a minor reduction in performance This would require of 6x Sample Buffer per channel loaded for a total of 18uL It is important to keep the ratio of the sample buffer to customer sample the same 14 How does one dispose of the chip after use Users should follow state local company academic regulations for disposal of biohazardous medical wastes 15 What happens if the ladder fails to be detected during a run If the software is unable to detect the real time ladder peaks it will automatically default to the Caliper method ladder This ladder is a virtual historical ladder based on the kit ladder provide
16. be compromised and should not be used e Top of chip was wet with buffer after run Ensure that the top of the chip is dry before beginning the run Buffer may form a liquid bridge in the openings to the buffer reservoirs during mixing of the dye Typically any meniscuses in these openings will break on their own within a few minutes Occasionally liquid in the meniscus will be transferred to the surface of the chip during a run Generally this has no impact but this occurrence can be eliminated by using a pipette tip to remove any meniscus of liquid in the openings to the reservoirs on the top surface of the chip prior to starting the run Caliper Life Sciences Page 15 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide e Inspect electrodes for buffer Use a lint free swab dampened with deionized water to rinse the electrodes if any buffer contamination is seen Allow electrodes to air dry before using instrument e Sieving material in chip appears cracked or dislodged after opening from foil packaging e Temperature of the chip may have exceeded optimal recommended temperature during shipment or storage Wide fluctuations in temperature may affect sieving material integrity e Foil seal over chip may have been damaged during shipment If a chip is not sealed properly fluctuations in pressure may affect sieving material integrit
17. d Caliper Life Sciences Page 20 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide 16 Can recover sample from the waste reservoir Yes DNA left in the waste reservoir can be recovered once a run has been completed If the DNA is to be recovered from the waste reservoir it is recommended that the user run an assay for the maximum allowable time extract and continue or exclude to ensure that the DNA of interest has entered the waste reservoir Standard purification kits can be used to purify DNA from the collected waste reservoir 17 see a scratch on the bottom of the chip Will this interfere with optical detection Minor scratches on the bottom surface of the chip will not interfere with optical detection 18 My background level of fluorescence seems high Can clean the optical detection area Yes See Cleaning LabChip XT in the user manual for a detailed description on how to clean the optical windows on the XT instrument 19 One channel seems to be running consistently faster than the other channels Will this affect my size selection oome variation between channels is normal and migration times are normalized by the lower marker in each channel Severe and consistent variations may indicate a problem with the instrument Run the instrument diagnostics and report any failures to Caliper Technical Support
18. de Introduction Applications oizing nucleic acid fragments is a time consuming but critical part of many library construction protocols Traditional gel based methods are notoriously inefficient and introduce variability that can negatively impact data quality For paired end and mate pair libraries accurate size selection is critical and improves assembly and facilitates the detection of structural variation Non sized libraries can be more difficult to assemble and often have errors in the detection of insertion and deletion events Accurate sizing is also critical for removing artifacts introduced during the library construction process such as adapter dimers Separating fragments containing library from no insert fragments significantly improves the amount of data obtained from the sequencing reaction The benefits of accurate and tight size selection include improved assembly of the sequenced data less non align reads and increased average read length The LabChip XT XTe fractionation system performs fast automated nucleic acid sizing accurately and reproducibly The XT XTe replaces the traditional gel method and tightly sizes up to four samples in less than 60 minutes Manual methods introduce run to run variability resulting in less accurate sizing and variable collection widths The LabChip XT XTe provides improved collection efficiency gt 50 and superior sensitivity detecting less than 25ng of sheared sample Fractionated sampl
19. ded into one channel is 1ug of sheared DNA fragmented to a distribution within the kit s specifications XT750 50 750bp XT300 25 500bp or 50ng of a single fragment double stranded DNA What happens if overload the chip with DNA Overloading a particular channel with more than 1ug of sheared DNA can result in poor sizing and or resolution Loading DNA beyond specifications may lead to under quantification of the total mass optical saturation and an electropherogram that is not representative of the actual population of input DNA What are the smallest and largest sizes that the XT can recover The XT DNA 750 300 kit can recover DNA sizes centered from 50bp to 1000bp 25bp to 500bp How can extraction width be measured For the Gaussian peaks that are typically extracted the standard deviation of the peak can be described as FWHM 0 425 where FWHM is the full width at the high signal maximum The CV can then be calculated as the standard deviation divided by the peak size CV 0 425 FWHWM size Caliper Life Sciences Page 17 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide 8 Whatis the minimum width that can be collected For the tightest size selection it is recommended a collection width of 5 be used Extraction widths of the collected material at the 5 collection width setting will be lt 10 CV fro
20. does not contain high amounts of salt This will reduce the stacking of DNA during separation The recommended sample diluent is EB or TE pH 8 0 8 5 e During the run the ladder peaks suddenly appear broad or slow down in migration e Ensure that there is a sufficient amount of buffer in the reservoirs e Ensure that the dye is mixed uniformly throughout the buffer waste reservoir Non uniformity across a reservoir may contribute to variation in sample migration within the separation channel e Check for buffer on the surface of the chip which may create a short between electrodes e Ensure that the sample diluent used in the ladder does not contain high amounts of salt which can affect DNA migration and stacking e Size selected material was too large or too small e Non homogeneity of dye within a reservoir can cause differences in migration It is important that the dye is mixed well in the waste reservoir e Ensure that the sample well has not been overloaded with DNA To achieve the specified sizing accuracy the sample load should not exceed 1000ng of sheared DNA as over sizing can occur with overloading e High amounts of salt can affect DNA migration and stacking which may lead to inaccurate sizing The sample is recommended to be in EB or TE pH 8 0 8 5 e Size selected material has shoulder on leading edge or trailing tail of peak when size selected material has been analyzed by LabChip microfluidic separation e Shoulders or
21. eared genomic DNA 50ng per fragment Minimum load capacity for optical detection 25ng sheared genomic DNA 0 5ng per fragment LabChip XT DNA 300 DNA size range 25 to 500 bp Minimum size distribution as expressed by CV 5 between 25 and 500 bp Maximum load capacity 1 ug of sheared genomic DNA 50ng per fragment Minimum load capacity for optical detection 25ng sheared genomic DNA 0 5ng per fragment Sample Recovery 50 90 6 Caliper Life Sciences Page 3 of 24 PN 450713 Rev 07 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Vh Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Kit Contents LabChip XT DNA 750 Assay Kit part number 760541 e XT DNA 1K Ladder yellow vial O e XT DNA 6x Sample Buffer green vial 9 XT DNA Collection Buffer red vial 9 e XT DNA Dye blue vial XT DNA Stacking Buffer pink purple vial e Store at 4 C LabChip XT DNA 750 Chip Kit 5 Chips e Store between 18 28 LabChip XT DNA 300 Assay Kit part number 760601 e XT DNA 500 Ladder yellow vial O e XT DNA 6x Sample Buffer green vial 9 e XT DNA Collection Buffer red vial 9 e XT DNA Dye blue vial O e XT DNA Stacking Buffer pink purple vial e Store at 4 C abChip XT DNA 300 Chip Kit 5 Chips e Store between 18 28 Additional Items Required abChip XT XTe instrument with LabChip XT software version 2 0 SP1 or greater may be downloaded from http w
22. es are delivered in a sequencing compatible buffer The XT improves laboratory efficiency and provides a level of sizing accuracy that is difficult to obtain using manual methods Data is displayed digitally and non fractionated sample can be recollected and used at another time Caliper Life Sciences Page 2 of 24 PN 450713 Rev 07 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 T Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Features Fast separation time separate up to 750bp in 30 minutes or less Eliminates slab gels and manual excision to size select DNA Automated sizing and quantitative information in a digital format Included reagent set provides predictable reproducible results Resulting sample is tightly sized and is delivered in a sequencing compatible buffer oize Selection may be performed using a software ladder thus maximizing throughput Each run can utilize 1 4 channels Channel Operation Modes Disabled Ladder eXtract and Stop eXtract and Continue eXcLuDe Hegion XT only Separation XT only eXtract and Pause or Skip Extraction XT only e Extraction Modes size fluorescence XT only peak start peak max XT only smear max XT only or collect on click Specifications LabChip XT DNA 750 DNA size range 50 to 750 bp Up to 1000bp with reduced resolution Minimum size distribution as expressed by CV 5 at 300 bp Maximum load capacity 1 ug of sh
23. igure 3A LabChip XT Electropherogram Figure 3B LabChip GX Electropherogram of collected material Example 4 Multiple Extractions across 400 600 bp Region eXtract and Pause mode XT 750 1 Channel 1 Caliper sizing ladder 2 Channel 2 100ng input select by size range otep 1 Extract and Pause 430bp 7 blue Step 2 Extract and Pause 500bp 7 red otep 3 Extract and Pause 568bp 6 brown Chip XT750 2011 05 23 03 25 13 Ladder Sizing Ladder 100 5000 300 400559 700 XTFractions 2011 05 23 04 23 04 50 Lb ux E Chip _XT750_ 2011 05 23 03 25 13 CH3 Sample 3 764 955 26 B5888888 Fluorescence aq a a o a a LL Fluorescence Rigned 88 ua m ids Size Bb e jii Figure 4A LabChip XT Electropherogram Figure 4B LabChip GX Electropherogram of collected material The green trace represents the pooled collections Caliper Life Sciences Page 11 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Calipe Y LabChip XT DNA Kit Assay User Guide Example 5 Discrete Extractions at 180 and 400 bp eXtract and Pause mode XT 750 1 Channel 1 Caliper sizing ladder 2 Channel 2 100ng input select by size range 180 400bp 5 blue red Chip81XT750_2011 05 23 04 11 28 Ladder Sizing Ladder pn jp 300 0020 400500 XIFractions 2011 05 23 04 54 41 8 E pu d a E Fluorescence Fluorescence 20 30 50 0
24. indows on the XT instrument are not blocked or contaminated with any potentially fluorescent material such as paper dust dyes reagents or materials that may block light Contaminants must be removed before starting a run e Dust can be removed with pressurized and filtered air If using a disposable pressurized air can hold the can upright to avoid spraying propellant onto the optics Usea lint free wipe or swab dampened with deionized water to rinse the chip locator e Allow all dampened surfaces to air dry e Do not use detergents or other cleaners as they may contain fluorescent dyes that interfere with the run e Sample may not have been properly loaded into the sample well If there are unused channels on the chip repeat ladder load on a different channel and if the problem persists repeat with a fresh vial of ladder Ensure that the sample diluent used with the DNA ladder 2uL 6x Sample buffer 2uL ladder sample diluent does not contain high amounts of salt This will reduce the stacking of DNA during separation The recommended sample diluent is TE or EB pH 8 0 8 5 Caliper Life Sciences Page 13 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Ladder peaks are too broad e Broad or non Gaussian ladder peaks are most commonly the result of poor sample loading Ensure that the DNA sample is no
25. ize selection and one for the ladder Use the Caliper method ladder which is a virtual historical ladder based on the kit ladder provided This allows the user to have four channels for size selection Use a custom ladder provided by the user custom ladder may be used as an alternative to the kit ladder when a user wants to have more peaks in a region of interest This allows the user to have three channels for size selection and one for the ladder The Sizing Table Tab contains the following settings Method Ladder selected the default Caliper supplied ladder is used to align the samples during the run Custom Ladder If selected the sizes in the Size Table as defined by the user are used For optimal sizing accuracy when using the size range extraction mode Caliper recommends using the Caliper supplied ladder Optically assisted extraction modes often do not require the use of the supplied ladder 12 If don t use all four channels during a run can I use the remaining channels later Yes but Caliper does not guarantee that the chip will comply with specifications of the kit when it is partially used It is critical that the chip remains sealed when not in use to prevent drying of the gel matrix For optimal performance Caliper recommends using all channels at once Caliper Life Sciences Page 18 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ife
26. le o Check to see that 20uL of collection buffer and stacking buffer sample have been added to their respective wells Collection buffer is required in all channels used even if no collection will take place o Checkto see that the surface of the chip and the underside of the instrument lid are free of buffer If any buffer is found remove buffer before beginning run again If it is observed that the reservoirs do not have sufficient levels of buffer to cover the chip electrodes the chip is defective please contact Technical Support for chip replacement o Note if directed by Caliper Technical Support it is possible to run the connectivity test separate from starting a chip run See the description of the Test Chip button on the Start Fractionation Window in the LabChip XT User Manual e Lower Marker LM was not detected e An internal standard LM is added to samples to assist in determining size and concentration of the sample The marker is also the first peak in the ladder The lower marker is contained in the XT DNA 6x Sample Buffer green vial o Ifthe lower marker is not detected the marker might have degraded over time due to exposure to light Repeat run again with fresh vial of 6x Sample Buffer o A second possibility is that insufficient amount of 6x Sample Buffer was added to the sample Ensure that the pipette is accurately dispensing 2uL of XT DNA 6x Sample Buffer e Ladder was not detected e Check that the optical w
27. le fractions 7 What happens when buffer capacity is exceeded The pH and current will begin to drift when the buffer capacity is exceeded eventually resulting in poor performance There is no sudden drop in performance when the warning is issued the limits are offered as a guideline that should give the most consistent results 8 How dol setup multiple extractions Refer to the LabChip XT Run File Guide 9 Whatare the recommended threshold settings for Peak Start and Peak Max for extracting small RNA library With some sample types such as dsDNA libraries containing sequences derived from small RNA fragments it is necessary to extract material that is very close in size to smaller unwanted material The Skip Extraction mode allows the user to identify and exclude the peak of smaller unwanted material containing no insert from the adjacent peak containing larger fragments of interest We recommend purifying the small RNA library prior to extraction Caliper Life Sciences Page 19 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide For most libraries in step 1 choose Skip Extraction for Operation define Search Region for 50 200bp choose Peak max for Trigger Mode and set Threshold for 5 5ORFU we recommend 25RFU lower thresholds may mistake baseline noise as a peak higher thresholds may miss a peak and set Collectio
28. m 50bp 750bp for the XT750 Assay and lt 7 5 CV from 25bp 500bp for the XT300 Assay Extractions above 750bp will have slightly wider extraction widths typically lt 15 CV Collection widths below 5 will decrease recovery with very little decrease in the size distribution of the collected material Based on the requirement of the experiment the user can optimize the collection width to ensure the appropriate balance between a tight size selection and optimal recovery 9 Whatis the maximum width that can be collected The collection width can be increased to 15 to increase the amount of material recovered Collection widths above 15 can result in over sizing with only small increases in the amount of material recovered Based on the requirement of the experiment the user can optimize the collection width to ensure the appropriate balance between a tight size selection and an optimal recovery 10 What are typical recovery rates of sample Typical recovery rates of DNA sample are 5096 9096 when using the eXtract and Stop or eXtract and Pause mode 11 Can load DNA smear in all four channels and size select without the use of a ladder Yes The Sizing Table Tab in the Start Fractionation Window specifies the ladder to use when aligning all the sample channels on the chip A user has three choices for a ladder Use the Caliper ladder included in kit in a channel on the chip This allows user to have three channels for s
29. m of collected material Example 2 Ladder reproducibility within one chip separation only mode 220ng of ladder in each channel XT 750 Migration Variation E T o 10 MES 40 o ps T FPaGEPIO01507200 E t B m P500 _ EP700 gt 850 1000 so n d 25 z o 10 20 Aiad Time gute 30 40 so 2 2 amp 20100721 850 5 sep chipZ LP4 2010 07 21 03 54 25 CH3 1K ladder 5 m EPSO 2 EP300 EMEP 700285071000 15 EP25 o i F us t E 10 20 Time rin 30 4c so 5 LM EPSD EP100 150 200 EP300 P400 psoo EP700 50 1000 o 4 1 iH t an 0 200 400 600 800 1000 1200 Fragment Size bp Figure 2A LabChip XT Electropherogram Figure 2B Migration Variation over three chips Caliper Life Sciences Page 10 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Calipe Y LabChip XT DNA Kit Assay User Guide Example 3 Advanced Extraction Modes 250ng of DNA XT 300 1 Channel 2 500ng input Step 1 Skip Extraction Peak Start Threshold at 150RF min Width at 30bp grey Step 2 Extract and Stop Peak start Threshold at 150RFU min Width at 10 red 2011 05 16 03 47 13 CH2 500ng XT 2011 05 19 11 47 58 C2 XT 3 3 1 5 117 a o a o n a LI a LL 30 40 4 6 10 20 4060100 200 40600000000 Aligned Time min Size BF F
30. mplete When used for multiple extractions the first step of the extraction is set to Skip Extraction typically using Peak Max as the extraction mode The software will automatically identify the peak within the Search Caliper Life Sciences Page 6 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Region and prevent the next step from triggering through the size range designated by the Collection Width The peak identified in the Skip Extraction step will be marked in grey on the electropherogram The second step typically Extract and Pause for the peak of interest may be set with an overlapping Search Region 5 For each channel select desired extraction mode using the radio buttons With the exception of Size Range the collection Width can be specified as the total number of base pairs BP a percent of the size at which extraction is centered or to be trigger by a Manual click a f Size Range The specified size range will be diverted to the chip collection well Size range can be specified on the Setup Tab as a Size a percent of the size or on the Advanced Tab as a specific start and end size in the Extraction Region text boxes The Setup Tab and Advanced Tab are synchronized to always specify the same settings Fluorescence Collection begins when the specified fluorescence Threshold in RFU is e
31. n Width for 25 30bp depending on width of the adapter dimer peak Using Peak Max for Step 1 will center the skip region at the peak and is less sensitive to peak fronting This is the recommended method Peak Start can be used if the falling edge of the peak to be skipped is not sharp enough for the software to identify a maximum Peak Max requires both positive and negative slope thresholds to be exceeded Peak Start only requires a positive slope threshold to be exceed Smear Max may also be used when the expected height of the peak to be skipped is known Increasing the skip collection width for Step 1 will delay the start of Step 2 and should reduce carryover of the unwanted peak For step 2 choose Extract and Pause for Operation define the Search Region for 50 200bp choose Peak Start for the Trigger Mode and set the Threshold for 10 200 we recommend 50RFU higher threshold might miss the peak and set Collection Width for 5 1096 depends on width of the library peak Using Peak start for Step 2 minimizes the amount of possible carryover of the unwanted peak Increasing width for Step 2 will increase the extraction time but keep the same start and may increase recovery 10 Is a separate chip test required before the actual run No it is not required but may be performed for troubleshooting purposes e g when instructed by Caliper Technical Support When running a separate chip test put running buffer in the in the sample
32. t being mixed with stacking buffer and that the sample is being loaded as described in step 12 of the section Prepare XT DNA Chip above the sample well not completely removed by the combs can also cause broad ladder peaks Ensure that all agarose has been removed from the sample wells without disrupting the gel in the channel See Chip Well Aspiration Using a Vacuum or Manual Pipette at the end of this guide Ensure that the stacking buffer pink vial was used in the sample well and not another reagent Without the stacking buffer the peaks will appear broad e Ensure that the sample diluent being used with the DNA ladder 2uL 6x Sample buffer 2uL ladder 8uL sample diluent does not contain high amounts of salt This will reduce the stacking of DNA during separation The recommended sample diluent is EB or TE pH 8 0 8 5 e Ladder has double peaks Sample may not have been properly loaded into the sample well or there may have been excessive gel in the sample well Ensure that all agarose has been removed from the sample wells without disrupting the gel in the channel See Chip Well Aspiration Using a Vacuum or Manual Pipette at the end of this guide If there are unused channels on the chip repeat ladder load on a different channel and if the problem persists repeat run again with fresh vial of ladder e Ensure that the sample diluent being used with the DNA ladder 2uL 6x Sample buffer 2uL ladder 8uL sample diluent
33. t is selected by clicking at the desired time You must click on the word CLICK in the Run Setup section on the left side of the LabChip XT Main Window A window displays the time until the collection starts This is the time until the selected sample size reaches the switch point where it is diverted into the collection well You can also choose to start collection immediately or cancel collection Based on the start size the software projects the median collection size and continues to collect material until stopped manually reached total number of BP or percent Collection Width specified in the text box Smear Max In Smear Max mode the software will search within the specified region for the point at which two consecutive conditions have been met 1 the signal has reached or surpassed the specified threshold signal RFU and 2 the signal is decreasing after averaging over three consecutive data points 6 For XT only click the Sizing Table Tab in the Run File Editor window a the default Caliper ladder select the Method Ladder option b To use a custom ladder by specifying the sizes of the peaks in the ladder select the Custom Ladder option and type the ladder peak sizes into the size column of the ladder table 7 Click on Output tab and confirm Data Path and input the File Prefix Project Name 8 Click the Save button and save the created run file to the desired directory Caliper Life Sciences Page 7 of 24 PN 45071
34. tails non Gaussian behavior can occur when samples are not loaded properly Ensure that the DNA sample is not being mixed with stacking buffer and that the sample is being loaded as described in step 12 of the section Prepare XT DNA Chip above Caliper Life Sciences Page 14 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide e Fluorescent signal is weak Secondary structure within the sample during separation can also lead to non uniformity of fractionated material To confirm that secondary structure within sample is the cause of non uniform peaks if feasible try heating a sample post extraction to 70 C for a few minutes snap cool by immersing sample in ice then reanalyze If DNA no 20 longer has shoulder or tail the irregular shape of the size selected material was an artifact of the analytical assay Figure 7 is 15 Check that the optical window on the XT instrument is not blocked or contaminated with any potentially Figure 7 Secondary structure of fractionated sample red fluorescent material such as paper dust dyes or ne reagents Contaminants must be removed before starting a run e Dust can be removed with pressurized and filtered air e Toclean spills use a lint free wipe or swab dampened with 7096 Isopropanol solution in deionized water to clean the lid and chip locator Run an optics diagnostic
35. ular Sample Wells O 11 Add 20uL of the Collection Buffer to round Collection Wells 9 Ensure that no bubbles remain in the well before proceeding to the next step Load the entire 12uL of the sample or prepared ladder into Sample Wells by layering under the Stacking Buffer Position the pipette so that the tip gently touches but is not sealed against the bottom of the well Pipette sample very slowly The goal is to place the sample at the bottom of the well and to avoid mixing the sample with the stacking buffer ADD COLLECTION amp STACKING BUFFER For best results start the XT run within 5 minutes of loading the samples into the wells of the chip Starting the LabChip XT 1 Place the chip in the LabChip XT instrument with the positioning feature in the upper left corner aligning with the corresponding pattern on the instrument 2 Close lid and select Instrument gt Start Run or move the mouse over the instrument icon and click on the green run arrow To import the settings from a previously saved run file click the Import Settings button at the bottom of the Start Fractionation window 4 Select the desired run file click the Open button and then click the Run button 5 SeelabChip XT User Manual or Run File Guide for more details on run parameter selection Caliper Life Sciences Page 8 of 24 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 h m Caliper
36. ww caliperls com support software downloads htm e Buffer for dilution of ladder EB 10mM Tris Cl pH 8 8 5 TE Buffer 10mM Tris Cl 1mM EDTA pH 8 8 5 e Microcentrifuge tubes for sample preparation Caliper Life Sciences Page 4 of 24 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT DNA Kit Assay User Guide Safety Warning amp Precautions WARNING 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 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 Dye vial contains DMSO Kit dye interacts with DNA and care should be taken to avoid inhalation and contact with skin and eyes Caliper Life Sciences Page 5 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Vh Wf Caliper ifeSciences abChip XT
37. xceeded Peak Start Collection begins at the start of a peak when the specified slope Threshold in RFU minute is exceeded Peak Max Centered on the first local maximum in the specified region The signal slope within the search region must exceed the specified slope threshold RFU min to arm this trigger The extraction is triggered when the signal has switched to a negative slope which meets or exceeds the specified threshold in magnitude If the end of the search region is reached after the trigger has been armed but before the negative slope threshold has been achieved the extraction will trigger at the end of the search region Collect On Click Collection is initiated by the user i LabChip XT software version 2 1 and later To select the highlighted region click on the word CLICK in the Run Setup section on the left side of the LabChip XT Main Window A dialog window will prompt you to confirm or cancel and the target region will continue to update until confirmed Note that for some width settings the target region may extend past the last visible data point on the graph In this case the highlighted region will end at the last visible data point and the dialog box will indicate at what time the target region ends When manual mode is selected in the width setting click on the word STOP in the Run Setup section to end the collection or exclusion li LabChip XT software version 2 0 SP3 and earlier The extraction trigger poin
38. y e affected channels should not be used e Chip leaks buffer from the bottom of the chip e Chip was damaged during handling or shipment contact Caliper Technical Support e Note that once the seal is removed the chip should not be inverted as buffer can flow out of the reservoir openings in the top of the chip Such leakage would be expected and does not indicate a defective chip e A buffer reservoir is empty after opening foil packaging e The chip is defective contact Caliper Technical Support e Sample well filled with buffer after comb removal or after excess sieving material was vacuumed from a well the sieving material in the channel is disrupted buffer can continuously flow into the sample or collection well from surrounding reservoirs This is a symptom of the sieving material having been compromised or dislodged in the channel and can lead to poor resolution lf the sample well fills with buffer only after removing of excess gel by vacuum take care not to form a strong vacuum seal at the bottom of the well thereby disrupting or possibly removing gel from the channel lf the sample well fills with buffer immediately after comb removal and refills after buffer is removed then the chip is defective contact Caliper Technical Support Caliper Life Sciences Page 16 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 1 877 522 2447 Ch Wf Caliper ifeSciences abChip XT
39. y in research conducted by the buyer whether the buyer is an academic or for profit entity including research services that do not involve the transfer of this product or its components or derivatives thereof The sale of this product is expressly conditioned on the buyer not using the product or its components 1 in manufacturing or in quality assurance or quality control 2 for therapeutic diagnostic or prophylactic purposes 3 to resell sell or otherwise transfer this product or its components to any third party whether or not such product or its components are resold for use in research or use for any use other than use in research or for any other commercial purpose Life Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a commercial product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product For information on purchasing a license to this product for purposes other than research contact Life Technologies Corporation Cell Analysis Business Unit Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0354 Copyright Caliper Life Sciences 2011 http www caliperLS com Caliper Life Sciences Page 24 of 24 PN 450713 Rev 08 68 Elm Street Hopkinton MA 01
Download Pdf Manuals
Related Search