Ampli1™ WGA Kit
Contents
1. cycler Otherwise the two steps will be done subsequently in the same thermal cycler storing the samples at 4 C This step is a pre preparation of a component for the reaction mix of step 3 for each different reagent calculate the volume required by multi plying the overall number of samples times the required volume for one sample Optional Preannealing Reaction Mix can be pre made and stored at 20 C However Preannealing Reaction Mix for all 50 samples cannot be made in a single 0 2 ml PCR tube as the total volume will exceed the limit of volume under temperature control in the thermal cycler Therefore it is advisable to divide Preannealing Reaction Mix into aliquots cor responding to the planned use eg if each Amp 1 WGA run tipically includes for 10 samples it is advisable to divide the Reaction Mix into 5 ali quots for 10 samples each Version 02 page 13 of 18 Ampli1 Whole Genome Amplification Procedure Tab 2B 1 Preparation of Preannealing Reaction Mix Volume per 1 Volume per 10 Hl esl Cap Color reactions ul reactions pl R1 Ampli1 white 0 5 5 0 Reaction Buffer 1 R4 Ampli1 Reagent A yellow 0 5 5 0 R5 Ampli1 Reagent 5 yellow 0 5 5 0 H2O Ampli1 M Water colorless 1 5 15 0 per reaction 3 0 30 0 Once the mix has been prepared briefly vortex and spin down in order to col lect all the reaction mix Do not add Preannealing Reaction Mix to the samples 2B 2 Incubate the preannealing reaction m2. reaction results No precipitation steps all preparatory steps are performed in one tube to avoid template loss Complete representation of the genome with fragments of about 0 2 2 kb Optimized amplification of all adapter ligated sequences Ampli1 WGA Kit Version 02 page 5 of 18 Ampli1 WGA Kit Application 8 Ampli1 WGA Kit Application The Ampli1 WGA Kit and procedure allows many different types of down stream analysis procedures such as Mutation detection by sequencing Mutation detection by pyrosequencing SNP detection Microsatellite or other PCR based genotyping analysis Metaphase CGH ArrayCGH Next Generation Sequencing Changes might be needed to adapt research protocols for the above tech niques to be compatible with the Ampli1 Whole Genome Amplification product output Please contact Silicon Biosystems Technical Support to check for compatibility with your research protocol An example is provided in Appendix A for illustration purposes only GU Amplii WGA uses a polymerase with proofreading activity with lower error rate 4 8X10 6 with respect to standard Taq DNA polymerases 9 Howto use the Amp i1 WGA product Ampli1 WGA Kit Quality Control to evaluate the quality of the Amp 1 WGA output product it is strongly advisable to run the Amp 1 QC Kit Silicon Biosystems Product code WG QC4 200 RWB This assay is a multiplex PCR of four markers indicative of the q
3. For research use only Not for use in e SEI Z H Z diagnostic procedures For n vitro use only Amplit WGA Kit Whole Genome Amplification for Single Cells USER MANUAL li Version 02 WG 001 050 RO2 50 reactions Store the kit at 20 C Visit www siliconbiosystems com for the most up to date version of this document P silicon biosystems A THE LIVING CELL COMPANY 1 KIL COTE E 3 2 St rage and Handlung eege 4 3 Intended Use amp Product Use Limitation ccsssssccsssssnssesssseseesssnneeesssneneeessenes 4 4 Safety MNIOrmatior diy 5i i lik Eege EENEG 4 5 Technical Assistance i yi d l mal silan alek k n Ek ka H k kak k kK ee du ke r kak u KER A R H Mu HU GE WAE 4 6 Additional Required Materials cccsssssssssscssssneesscessssnnnneeeesseesenneeeeseeessneeneeseress 5 7 Ampli1 WGA Kit Description cscsssssssssnssnsensensessesteseestesnesneeneeneensenseseesoeses 5 8 Ampli1 WGA Kit Application c cscsssssssssssssnsenensesessestesteneesnesneencenseneessesneses 6 9 How to use the Ampli1 WGA product c sssscsnsessessesnsessesnseneeseeeneesneensennesneeses 6 10 Sample Specifications ccssssssssssssssssssnnssssssnneessssnnnessssennessssennnessesennessssseneesness 8 11 Things to Do Before Starting sccsssessssseessnseessnneeessneeessnneeeseneeessneeeessnneessnneeesenes 9 12 Amp
4. ata sheets MSDS MSDS of all Silicon Biosystems kits and components are available online at http www siliconbiousa com 5 Technical Assistance For technical assistance and additional information please refer to Silicon Bio systems Technical Support Molecular Biology Department e mail amplil support siliconbiosystems com Telephone number 39 051 40 71 300 Ampli1 WGA Kit Version 02 page 4 of 18 Additional Required Materials 6 Additional Required Materials Thermal Cycler e g Applied Biosystems GeneAmp 9700 or superior Dedicated pipette set PCR microcentrifuge tube 0 2 ml Recommended MicroAmp Reaction tube with cap 0 2 ml Applied Biosystems Part No N801 0612 Barrier tips Mini Centrifuge suitable for PCR tubes Laminar flow hood e 20 C Storage Freezer Vortex 7 Ampli1 WGA Kit Description The Ampiii Whole Genome Amplification kit has been specifically developed and optimized for the amplification of the total DNA content of a single cell The Ampli1 WGA procedure is based on a ligation mediated PCR following a site specific DNA digestion The output of an Ampli1 WGA procedure is a library of highly concentrated DNA which can be employed for further targeted genetic research analysis The main features of the Ampii1 Whole Genome Amplification kit are Comprehensive and homogenous amplification of the whole genome isolated from a single cell Robust and reproducible
5. bes tube racks 0 2 ml PCR tubes compatible centrifuge vortex lab coats 20 C freezer etc Use barrier tips Eppendorf Dualfilter PCR clean sterile are suggested Once Primary PCR Reaction thermal cycling program has finished remove the tubes from the thermal cycler and store them in a 20 C freezer in a sep arate lab space dedicated to downstream analysis of amplified products Perform each type of downstream analysis e g PCR sequencing mCGH etc in a separate lab with dedicated equipment and materials this step is the most important aspect in order to avoid carryover of amplified DNA 2 Control Samples It is recommended to process the following controls along with samples in each run of an Ampli1 Whole Genome Amplification procedure No cell control 1 ul of Amp i1 Water Positive Control prepare a positive control by adding in the positive control tube 1 ul of DNA 1ng ul concentrated In order to avoid cross contamina tions it is suggested to process the positive control as last sample of each step Remember to take into account the no cell and positive control samples when setting up the correct volume of each reaction mix For example to amplify 8 samples prepare reaction mix for 10 samples to accomodate the no cell and positive controls 3 Pipetting Tips All pipetting must be carried out under the dedicated laminar flow hood The Ampli1 WGA procedure requires working with very small vol
6. d start the run Tab 3 2 Thermal incubation profile of Ligation Reaction Temperature C Hold Volume pl 15 1 hour 10 15 o0 Optional Ligation Reaction may be extended to an overnight incubation Remove all the reaction tubes put them in a microtube rack and store the samples at 4 C while preparing mix for next step Do Not Freeze the samples Directly proceed with Step 4 Ampli1 WGA Kit Version 02 page 15 of 18 Step 4 Primary PCR Ampli1 WGA Kit Ampli1 Whole Genome Amplification Procedure 4 1 Prepare Primary PCR Reaction Mix according to the protocol in Table 4 1 Tab 4 1 Preparation of Primary PCR Reaction Mix Volume per 1 Volume per 10 Vial Label Cap Color reactions wl reactions pl R7 Amplii purple 3 0 30 0 Reaction Buffer 7 R8 Ampli1 Reagent 8 purple 2 0 20 0 E4 Ampli1 Enzyme A purple 1 0 10 0 H2O Ampiii Water colorless 34 0 340 0 per reaction 40 0 400 0 4 2 Add 40 ul of Primary PCR Reaction Mix to each sample Pipette 40 ul of Primary PCR Reaction Mix it onto the wall of the tube above the other liquid already present 10 ul but without touching it Final volume 50 ul 4 3 Incubate the Primary PCR Reaction Mix according to Table 4 2 Briefly spin all the sample tubes Put samples in the thermal cycler and start the run as described in table 4 2 Tab 4 2 Thermal incubation profile of Primary PCR Reaction Cycle Temperature Additional time Volume Numbers C Hold an
7. d temperature yl 68 3 minutes 14 94 40 sec D7 30 sec 68 1 30 min sec 1 sec cycle 8 94 40 sec D 30 sec 1 C cycle 50 68 1 45 min sec 1 sec cycle 22 94 40 sec 65 30 sec 68 1 53 min sec 1 sec cycle 1 68 3 40 min sec 4 CO Notes 1 sec cycle 1 C cycle Store the samples at 20 C Version 02 page 16 of 18 Patent amp Trademark Information GU It is crucial to follow the thermal amplification profile indicated for the Pri mary PCR as it guarantees a good amplification yield for longer DNA fragments Please refer to the Thermal Cycler User Manual to correctly set the amplification parameters 13 Patent amp Trademark Information Use of this product is covered by US patent No 6 673 541 and corresponding patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research Silicon Biosystems SpA products may not be transferred to third parties resold modified for resale used to manufacture commercial products without written approval of Silicon Biosystems SpA Amplii is a trademark of Silicon Biosystems SpA or its subsidiaries which may be registered in certain jurisdictions Other brands and product names are trademarks of their respective holders 14 Warranty Ampli1 WGA Kit This product is warranted only to be free
8. e in Next Generation Sequencing 1 Amplicon based methods AmpliSegq or similar can be directly applied to the WGA DNA obtained from the procedure provided no Amplit restric tion site TTAA is present in the amplicon Appendix A 2 Capture based methods digestion of WGA adaptors is recommended before sequencing library preparation in order to avoid unwanted unspe cific product capture Please contact amplil support siliconbiosystems com to obtain latest version of the removal protocol 3 Whole Genome sequencing a sonication of the WGA product after WGA adaptor removal and before sequencing is advised to reduce size of larger WGA fragments Version 02 page 7 of 18 Sample Specifications 10 Sample Specifications Ampli1 WGA Kit The Ampli1 WGA procedure is designed to work with an input sample of one single cell in 1 pl of PBS1X The kit also can be used to amplify the DNA content from samples containing higher number of cells up to 1000 or DNA e g from tissue or FFPE samples resuspended in 1 ul of PBS1X Although best results are obtained with live cells the Ampli1 WGA Kit allows whole genome amplification from single fixed or fixed and permeabilized cells As an example good results have been obtained with the following Paraformaldehyde 1 2 PFA 10 20 at RT Single cells isolated from blood samples collected in CellSave tubes and processed with Veridex CellSearch Samples process
9. e wall of the tube above the other liquid already present 1 ul but without touching it Final volume at this point 3 ul 1 3 Incubate all samples according to Table 1 2 Briefly spin down all the sample tubes prior to placing them in the thermal cycler Ampli1 WGA Kit Version 02 page 11 of 18 Step 2A DNA Digestion Ampli1 WGA Kit Ampli1 Whole Genome Amplification Procedure Tab 1 2 Thermal incubation profile of Lysis Reaction Temperature C Hold Volume pl 42 45 minutes 65 30 minutes 3 80 15 minutes 4 Co Once the thermal cycler has reached 4 C remove all the reaction tubes put them in a microtube rack and store the samples at 4 C while preparing mix for next step Optional for user convenience the lysis step incubation at 42 C can be extended up to an overnight incubation For the success of the entire procedure it is crucial to run the enzyme inacti vation steps 30 minutes at 65 C and 15 minutes at 80 C Those steps must be kept also if an overnight lysis step at 42 C is performed Do Not Freeze the samples Directly proceed with Step 2a 2A 1 Prepare Digestion Reaction Mix according to Table 2A 1 Prepare the Digestion Reaction Mix as follows for the overall number of samples by calculating the total volume needed as described 10 reac tions is an example Tab 2A 1 Preparation of Digestion Reaction Mix Vial Label Cap Color 2 peri OUTS pee reactions ul reactions pl R1 Am
10. ed with Inside Stain Inside Fix Inside Perm from Milte nyi Biotec GmbH Cell staining with antibodies conjugated with fluorophores does not affect the yield of an Ampii1 WGA amplification procedure Nuclei staining might negatively impact the yield staining with Hoechst 33342 Sigma Aldrich cat B2261 final staining concentration 1 ug ml 5 10 at RT is a suitable working condition Amplii Whole Genome Amplification Kit is also suitable for the amplifica tion of DNA from single sperm cells In this case it is mandatory to add 1 ul of DTT 100 mM in the lysis reaction calculated for 10 samples the volume of water should be decreased from 12 8 to 11 8 yl to reach a final concentration of 5 mM DTT in 20 wl For more than 10 samples adjust all reagent volumes Version 02 page 8 of 18 Things to Do Before Starting 11 Things to Do Before Starting Ampli1 WGA Kit 1 Working Area Organization The Amplit WGA Kit has been optimized to enable amplification of DNA content from one single cell as such it is a powerful tool to amplify nucleic acid In order to prevent any contamination due to carryover of amplified DNA it is strongly recommended to Dedicate a separate laboratory or at least a separate working space to sin gle cell amplification and organize it with dedicated materials such as lami nar flow hood thermal cycler pipettes pipette tips PCR 0 2 ml micro centrifuge tubes 1 5 ml micro centrifuge tu
11. from defects in workmanship and material at the time of delivery to the customer Silicon Biosystems SpA makes no warranty or representation either expressed or implied with respect to the fitness of a product for a particular purpose There are no warranties expressed or implied which extend beyond the Technical Specifications of the products Silicon Biosystems SpA s liability is limited to either replacement of the products or refund of the purchase price Silicon Biosystems SpA is not lia ble for any property damage personal injury or economic loss caused by the product Notice to Buyer User Information presented herein is accurate and reliable to the best of our knowledge and belief but is not guaranteed to be so Nothing herein is to be construed as recommending any practice or any product in vio lation of any patent or in violation of any law or regulation It is the user s responsibility to determine for himself or herself the suitability of any material and or procedure for a specific purpose and to adopt such safety precautions as may be necessary Version 02 page 17 of 18 Appendix A 15 Appendix A An example of The library of fragments generated through the Ampiii WGA procedures use of Amp i1 originates from DNA digested as follows WGA output in 5 TYTAA 3 PCR downstream 3 AATAT _ 5 research assays The specific action of the Digestion Enzyme makes it possible to determine the exact sequence of Amp
12. i1 amplification products around any target region Primer design Designing target specific PCR to amplify and analyze one target sequence in the Ampli1 WGA amplification product requires specific considerations 1 Identify the target of the downstream assay sequence mutation micro satellite etc 2 Download the DNA sequence containing the target 3 Determine where the flanking restriction sites are A Do not use mRNA sequence data as the flanking restriction sites could reside in introns 5 Extract the sequence of the WGA Amplicon that will contain the target 6 Design the downstream assay considering the WGA Amplicon gener ated 7 Do not design primers that overlap digestion sites e MOOG 3 Ge Forward Target sequence Reverse RR Digestion Primer Primers Digestion sequence sequence Fig 2 Primers design for PCR as downstream analysis Verification of primer pairs 1 Download from the database the sequence target encompassed by the primer pairs it is necessary to work on the DNA sequence as in the mRNA sequence some digestion sites could be hidden due to splicing 2 Verify that the target sequence of the primers does not include the digestion motif taking into account possible degenerate base variants if present 3 Do not design primers that overlap digestion sites Ampli1 WGA Kit Version 02 page 18 of 18 0514 06491600001
13. ix according to Table 2B 2 Put the Preannealing Reaction Mix in the thermal cycler and start the run as follows Tab 2B 2 Thermal incubation profile of Preannealing Reaction Cycle Numbers Temperature PC Hold Volume pl 1 65 1 minutes d 1 minutes Samples x 3 ul 15 1 minutes 1 15 Ka start at 65 C for 1 min incubation and then reduce for 1 C per cycle incubating for 1 minute for each temperature until 15 C GU it is crucial to follow exactly the thermal amplification profile for the Preaannealing reaction Please refer to the Thermal Cycler User Manual to set the correct parameters Ampli1 WGA Kit Version 02 page 14 of 18 Ampli1 Whole Genome Amplification Procedure Step 3 Ligation 3 1 Prepare Ligation Reaction Mix according to the protocol in Table 3 1 Tab 3 1 Preparation of Ligation Reaction Mix Volume per 1 Volume per 10 Vial Label Cap Color reactions wl reactions pl Preannealing 3 0 30 0 reaction R6 Amplii Reagent 6 green 1 0 10 0 E3 Amplii Enzyme 3 green 1 0 10 0 per reaction 5 0 50 0 3 2 Add 5 ulof Ligation Reaction Mix to each sample Pipette 5 ul of Ligation Reaction Mix it onto the wall of the tube above the other liquid already present 5 ul but without touching it Final volume at this point 10 ul Briefly spin the samples tube and put them back in a microtube rack 3 3 Incubate the Ligation reaction mix according to Table 3 2 Put all the samples in the thermal cycler an
14. ix and incubate and incubate amplify Total 1h 40 20 1h 10 3h 30 6h 40 1h 15 STEP 2B Ke Preannealing Prepare Preannealing M Reaction Mix and incubate Fig 1 Ampiii Whole Genome Amplification Procedure 2 Ampilii WGA procedure and reaction Before starting make sure that all the samples meet the requirements described in section 9 Sample Specification and that the working area is properly equipped Amplii WGA Kit Version 02 page 10 of 18 Ampli1 Whole Genome Amplification Procedure Step 1 Cell Lysis 1 1 Prepare Lysis Reaction Mix according to Table 1 1 lt is recommended to prepare a Lysis Reaction Mix for at least 10 reactions even if fewer than 10 samples will be run excess reagents for the Lysis Reaction Mix have been included in the Amp i1 WGA Kit To prepare a larger amount of Lysis Reaction Mix increase all reagent volumes propor tionally Tab 1 1 Preparation of Lysis Reaction Mix Volume per 10 Vial Label Cap Color reactions pl R1 Ampli1 white 2 0 Reaction Buffer 1 R2 Ampli1 Reagent 2 blue 1 3 R3 Ampli1 Reagent 3 blue 1 3 E1 Ampli1 Enzyme 1 blue 2 6 H O Ampli1 M Water colorless 12 8 20 0 per reaction 2 0 Once the Lysis Reaction Mix has been prepared briefly vortex and spin down in order to collect all the reaction mix at the bottom of the tube 1 2 Add 2 ul of Lysis Reaction Mix to each sample Pipette 2 ul of Lysis Reaction Mix onto th
15. li1 Whole Genome Amplification Procedure s scsssssessseseeenees 10 Step 1 Cell Lysis 11 Step 2A DNA Digestion 12 Step 2B Preannealing 13 Step 3 Ligation 15 Step 4 Primary PCR 16 13 Patent amp Trademark Information cssssssccssesssesssnnsecsssnseeeesesnneeessesneeesssnnneeess 17 14 Waranty eege ee 17 15 ADDOM XP kar s d d sael EEN 18 An example of use of Amp i1 WGA output in PCR downstream research assays 18 Amplii WGA Kit Version 02 page 2 of 18 1 Kit Contents Ampli1 WGA Kit Reagents Enzymes Vial R1 R2 R3 R4 R5 R6 R7 R8 H20 E1 E2 E3 E4 Label Reaction Buffer 1 Reagent 2 Reagent 3 Reagent 4 Reagent 5 Reagent 6 Reaction Buffer 7 Reagent 8 Water Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 Version 02 Cap Color white blue blue yellow yellow green purple purple colorless blue black green purple Kit Contents Contents 1 vial 90 wl 1 vial 50 ul 1 vial 50 wl 1 vial 35 wl 1 vial 35 wl 1 vial 70 wl 1 vial 1 000 wl 1 vial 200 wl 3 vial 1 000 wl 1 vial 30 wl 1 vial 15 wl 1 vial 60 wl 1 vial 70 wl page 3 of 18 Storage and Handling 2 Storage and Handling Store the Amp i1 WGA Kit at 20 C ship at 20 C Transfer Enzyme 1 2 3 4 tubes to ice just prior to use Other kit components should be thawed on ice and briefly vortexed before use When stored and handled unde
16. pli white 0 2 2 0 Reaction Buffer 1 E2 Amplii Enzyme 2 black 0 2 2 0 H2O Ampli Water colorless 1 6 16 0 per reaction 2 0 20 0 Once the mix has been prepared briefly vortex and spin it down in order to collect all the reaction mix 2A 2 Add 2 ul of Digestion Reaction Mix to each sample Pipette 2 ul of Digestion Reaction Mix onto the wall of the tube above the other liquid already present 3 ul but without touching it Final volume at this point 5 ul Briefly spin down the samples tube and put them back in a microtube rack Version 02 page 12 of 18 Step 2B Preannealing Ampli1 WGA Kit Ampli1 Whole Genome Amplification Procedure 2A 3 Incubate all samples according to Table 2A 2 Put all the samples in the thermal cycler and start the run as follows Tab 2A 2 Thermal incubation profile of Digestion Reaction Temperature C Hold Volume pl 37 5 minutes 65 5 minutes 5 Bho Once the thermal cycler has reached 4 C remove all the reaction tubes place them in a microtube rack and store the samples at 4 C while preparing the mix for next step Do Not Freeze the samples Directly proceed with Step 2B or if this step has already been done in a separate thermal cycler proceed with step 3 2B 1 Prepare Preannealing Reaction Mix according to Table 2B 1 This step could be done in parallel with step 1 advisable and or step 2A by using a different thermal cycler or a different plate in the same thermal
17. quired Please contact amplil support siliconbiosystems com for Ampiii WGA Reamplification protocol No cell Control the no cell control each from Amp i1 WGA run should be analyzed in downstream assays such as Amp 1 QC or Ampli1 Gene spe cific Kits A negative assay result from the no cell control confirms the absence of contamination in the WGA workflow Other evaluation methods may not be suitable for the Amp WGA no cell control some amplification of non human DNA background originating from the bacterial DNA present in the enzymes See chapter 3 of this manual may occur This will negatively affect quantitation methodologies and result in a smear on an agarose gel similar to what is typically obtained for the samples with cells For this reason running agarose gel to evaluate Amp 1 WGA yield is NOT recommended Use in PCR the Ampli1 WGA products can be directly used in PCR without dilution and or purification In standard amplification conditions 20 wl of reaction volume with 0 5 uM final concentration of each primer 2 ul of Ampli1 WGA product shall be used Lower or higher input could generate amplification drop out and or unspecific amplification Use of kits from the Ampli1 product line Silicon Biosystems for the amplification of cancer related mutations such as KRAS BRAF PIK3CA EGFR ALK is strongly recommended to take advantage of the pre validated and optimized reagents and conditions Us
18. r these condi tions the kit components are stable through the expiration date specified Handle and store reagents with the appropriate attention and care and set up reaction according to good laboratory practices for PCR Silicon Biosystems SpA recommends that the buyer and other persons using this product follow the Guidelines for Research involving Recombinant DNA Molecules NIH guidelines Federal Register July 5 1994 59 FR 34496 and any amendments thereto Silicon Biosystems SpA disclaims any and all responsibility for any injury or damage which may be caused by the failure of the buyer or any other person to follow said guidelines 3 Intended Use amp Product Use Limitation The Amplii Whole Genome Amplification Kit is intended for research use only The Amp i1 Whole Genome Amplification Kit is for in vitro use only No claim or representation is made for an intended use to provide information for the diagnosis prevention or treatment of a disease It is normal that some background of bacterial DNA will be present in the Amplii product at the end of the reaction even in no template controls Ampli1 WGA should not be used for bacterial samples The Amp i1 WGA Kit is not recommended for downstream analysis with BAC Array 4 Safety Information When working with chemicals always wear suitable lab coat disposable gloves and protective goggles For more information please consult the appro priate material safety d
19. uality of the DNA library obtained and predictive of the success rate of further downstream assays Please refer to the Amp 1 QC Kit user manual for further specification Purification Amp i1 WGA output product does not require purification for most downstream applications eg PCR sequencing If purification is needed it is advisable to use Agencourt AMPure XP kit Part Number A63880 Beckman Coulter which minimizes template loss Purification with this kit further eliminates residual unligated primers left in solution at the end of the WGA reaction Version 02 page 6 of 18 Ampli1 WGA Kit How to use the Ampli1 WGA product Quantitation for Amp ii WGA output product quantification Qubit Fluo rometer Life Technologies with the corresponding Qubit dsDNA BR Assay Kit ref Q32850 is recommended This method quantifies all the dou ble stranded DNA produced and present in the Amp ii WGA output products NOTE WGA primers and adaptors unligated to a fragment but left in the reaction after the WGA Primary PCR are also double stranded and they will be included in the overall quantitation of the sample In order to have a more reliable quantitation of the Ampli1 WGA target DNA it is suggested to per form a purification step prior to the quantitation Reamplification protocol 1 ul of Ampli1TM WGA product can be reamplified to obtain 50 ul of reamplified WGA DNA for further downstream analysis if re
20. umes to avoid loss of materials it is recommended to proceed as follow All the reactions described in the Ampli1 WGA procedure take place in the same tube in which the single cell has been originally isolated for that rea son it is important to carefully dispense the appropriate volume for each reaction without disturbing the liquid already present in the tube in order to avoid inadvertently removing the cell Add the required volume by pipetting the fluid directly onto the wall of the tube without disturbing the fluid already present in the tube Always collect all the liquid by a short centrifuge spin after adding reaction mix and before putting the samples in the thermal cycler Version 02 page 9 of 18 Ampli1 Whole Genome Amplification Procedure 12 Ampli1 Whole Genome Amplification Procedure 1 Ampli1 WGA procedure overview All the reactions required for Amp iii Whole Genome Amplification proce dure take place in the same tube in which the single cell has been isolated starting from 1 pl of PBS 1X Therefore all the reaction mixes will be subse quently added to that same tube as shown in Fig 1 STEP 1 STEP 2A STEP 3 STEP 4 Cell Lysis Add DNA Digestion Ligation Add Primary PCR ge y Lysis Reaction f d Add Digestion 2 ligation Ie Add Primary 12 Mix to each Zi Reaction Mix to Reaction mix lt j PCR Reaction sample and the same tube and incubate M
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