Ampli1™ WGA Kit
Contents
1. eere cree sn ennenntnnnntnnnnan 14 13 Warranty 15 LM APpPendix A 16 An example of use of Ampli1 WGA output in PCR downstream research assays 16 Ampli1 WGA Kit Version 01 page 0 of 16 1 Kit Contents Ampli1 WGA Kit Kit Contents Vial Label Cap Color Contents R1 Reaction Buffer 1 white 1 vial 90 ul R2 Reagent 2 blue 1 vial 50 ul R3 Reagent 3 blue 1 vial 50 ul g R4 Reagent 4 yellow 1 vial 35 ul S R5 Reagent 5 yellow 1 vial 35 ul R6 Reagent 6 green 1 vial 70 wl R7 Reaction Buffer 7 purple 1 vial 1 000 ul R8 Reagent 8 purple 1 vial 200 pl H20 Water colorless 3 vial 1 000 ul E1 Enzyme 1 blue 1 vial 30 ul 2 E2 Enzyme 2 black 1 vial 15 ul i3 E3 Enzyme 3 green 1 vial 60 ul DI E4 Enzyme A purple 1 vial 70 ul Version 01 page 3 of 16 Storage and Handling 2 Storage and Handling Store the Ampli1 WGA Kit at 20 C ship at 20 C Transfer Enzymes 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 under these condi tions the kit components are stable through the expiration date specified Handle and store reagents with the appropriate attention and care and setup reaction according to good laboratory practices for PCR Silicon Biosystems SpA recommends that the buyer and other persons using this product fo2. 01 page 5 of 16 Ampli1 WGA Kit Application 8 Ampli1 WGA Kit Application The Ampli1 WGA Kit and procedure allows many different types of down stream research procedures such as Mutation detection by sequencing Mutation detection by pyrosequencing SNPs detection Microsatellite or other PCR based genotyping Analysis Metaphase CGH Changes might be needed to adapt research protocols for the above tech niques to be compatible with the Amp i1TV Whole Genome Amplification product output Please enquire with Silicon Biosystems Technical Support to check for compatibility with your research protocol An example is provided in Appendix A for illustration purposes only 9 Sample Specifications Ampli WGA Kit The Ampli1 WGA procedure is designed to work with an input sample of one single cell in 1 ul of PBS1X The kit also can be used to amplify the DNA content from samples containing higher number of cells or DNA in 1 pl of PBS1X Although best results are obtained with live cells the Amplii WGA Kit allows the whole genome amplification also 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 processed with Inside Stain Inside Fix Inside Perm from Milte nyi Biot
3. 3 Incubate the Ligation reaction mix according to Table 3 2 Put all the samples in the thermal cycler and start the run Tab 3 2 Thermal incubation profile of Ligation Reaction Temperature C Hold Volume pl 15 over night 12 h 10 Ligation Reaction requires over night incubation Remove all the reaction tubes put them in a microtube rack and store the samples at 4 C while pre paring mix for next step A Do Not Freeze the samples Directly proceed with Step 4 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 po eis Cap Color reactions yl reactions pl R7 Ampli1 purple 3 0 30 0 Reaction Buffer 7 R8 Ampli1 Reagent8 purple 2 0 20 0 E4 Ampli1 Enzyme A purple 1 0 10 0 H2O Ampli 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 A Pipette 40ul of Primary PCR Reaction Mix delivering it onto the wall of the tube above the other liquid already present 10 pl but without touching it Final volume 50 ul 4 3 Incubate the Primary PCR Reaction Mix according to Table 4 2 Briefly spin down all the sample tubes Put all the samples in the thermal cycler and start the run as described in table 4 2 Version 01 page 13 of 16 Patent amp Trademark Information Tab 4 2 Thermal incubation profile of Primary PCR Reaction Cycle T
4. For research use only Not for use in diagnostic procedures For in vitro use only Ampli WGA Kit Whole Genome Amplification for Single Cells USER MANUAL l3 Version 01 WG 000 050 RO1 50 reactions Store the kit at 20 C Visit www siliconbiosystems com for the most up to date version of this document silicon biosystems THE LIVING y COMPANY H 1 Kit Contents enun LLL i el ienaa 3 2 Storage and Handling csccseseeeeeeesesseeenesenseeeneneneceenenenseeeeenensenenenenecneneneneeeeeenenseees 4 3 Intended Use amp Product Use Limitation eee 4 4 Safety Information eere eere e eere seen enata sn snsm ana tn sn sm uasa snam ata sa snam assa snam 4 5 Technical Assistance seess ege A 6 Additional Required Materials ssssssssseeeenssseeeessnenseereenenseenensneeneneneneceenenensees 5 7 Ampli1 WGA Kit Description see 5 8 Ampli1 WGA Kit Application Ee 6 9 Sample Specifications aeree e eere eren en ese snsn ana tntn amata tn sn ennnen annen 6 10 Things to Do Before Starting eee eres eeee eren tese snae tntns nata tnsn aman 7 11 Ampli1 Whole Genome Amplification Procedure eres 8 Step 1 Cell Lysis 9 Step 2A DNA Digestion 10 Step 2B Preannealing 11 Step 3 Ligation 12 Step 4 Primary PCR 13 12 Patent amp Trademark Information
5. ec GmbH Cell staining with antibodies conjugated with fluorophores does not affect the yield of an Amplit WGA amplification procedure Nuclei staining might negatively impact yield staining with Hoechst 33342 Sigma Aldrich cat B2261 final staining concentration 1 pg ml 5 10 at RT is a suitable working condition Version 01 page 6 of 16 Things to Do Before Starting 10 Things to Do Before Starting Ampli WGA Kit 1 Working Area Organization The Ampli WGA Kit is a powerful tool to amplify nucleic acid since it enables the amplification of the DNA content from one single cell In order to prevent any contamination due to amplified DNA carryover 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 pipette pipette tips PCR 0 2 ml micro centrifuge tubes 1 5 ml micro centrifuge tubes 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 take the tube off the thermal cycler and store them in a 20 C dedicated to the down stream analysis in a separate lab Perform each type of downstream analysis e g PCR sequencing mCGH etc in a separate lab with separate materials t
6. emperature Additional time Volume Numbers C rud and temperature pl 68 3 minutes 14 94 40 sec 57 30 sec 68 1 30 min sec 1 sec cycle 8 94 40 sec 57 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 Notes 1 sec cycle 1 C cycle A Store the samples at 20 C 12 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 Ampli 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 Ampli1 WGA Kit Version 01 page 14 of 16 13 Warranty Ampli WGA Kit Warranty This product is warranted only to be free 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 prod
7. his step is the most important aspect to care for in order to avoid carryover of amplified DNA to single cells samples 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 Ampli1 Water Positive Control prepare a positive control by adding in the positive control tube 1 ul of DNA 1ng pul 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 consider also 1 no cell control 1 positive control prepare reaction mix for 10 samples 3 Pipetting Tips All pipetting must the carried out under the dedicated laminar flow hood The Ampli WGA procedure requires working with very small volumes 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 with the pipette tip in order to avoid to inadvertently removing the cell Just add the required volume by pipetting
8. llow 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 Ampli1 Whole Genome Amplification Kit is intended for research use only The Ampli 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 Ampli product at the end of the reaction even in no template controls Ampli WGA should not be used for bacterial samples The Ampli 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 data sheets MSDSs MSDS of each Silicon Biosystems kit and components are available online at http www siliconbiosystems com mSDS page 5 Technical Assistance For technical assistance and additional information please refer to Silicon Bio systems Technical Support Molecular Biology Department e mail amplil suppor
9. ng 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 3 hours 65 5 minutes 5 4 oo Once the thermal cycler has reached 4 C remove all the reaction tubes plac ing them in a microtube rack and store the samples at 4 C while preparing the mix for next step A 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 the protocol in Table 2B 1 This step could be done in parallel with step 2A by using a different thermal cycler or a different plate in the same thermal cycler Otherwise the two steps will be done subsequently in the same thermal cycler storing the samples at 4 C A This step is a pre preparation of the reaction mix of the step 3 calculate the needed volume by calculating the overall number of samples multi plied by the required volume for one sample for each different reagent Tab 2B 1 Preparation of Preannealing Reaction Mix Volume per 1 Volume per 10 KI De Gp Gaor reactions I reactions pl R1 Ampli1 white 0 5 5 0 Reaction Buffer 1 R4 Ampli1 Reagent A yellow 0 5 5 0 R5 Ampli1 Reagent5 yellow 0 5 5 0 H2O Ampli Water colorless 1 5 15 0 per reaction 3 0 30 0 Once the mix has been prepared briefly vor
10. nsiderations 1 Identify the target of the PCR downstream assay sequence mutation microsatellite 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 just considering the WGA Amplicon gen erated 7 Do not design primers that overlap digestion sites s LLE Ss Digestion eeng Target sequence penis 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 also the possible degenerate base variants if present 3 Do not design primers that overlap digestion sites Ampli1 WGA Kit Version 01 page 16 of 16 0611 064915960010
11. of Lysis Reaction Temperature C Hold Volume pl 42 15 hours 80 10 minutes 3 4 o0 The Lysis Reaction requires an over night incubation 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 A Do Not Freeze the samples Directly proceed with Step 2a 2A 1 Prepare Digestion Reaction Mix according to the protocol in Table 2A 1 A 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 KEE reactions ul reactions pl RI Ampli1 white 0 2 2 0 Reaction Buffer 1 E2 Ampli1 Enzyme 2 black 0 2 2 0 H20 Ampli1 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 2A2 Add 2 yl of Digestion Reaction Mix to each sample A Pipette 2 ul of Digestion Reaction Mix delivering it onto the wall of the tube above the other liquid already present 3 ul but without touching it Final volume at this point 5 pl A Briefly spin down the samples tube and put them back in a microtube rack Version 01 page 10 of 16 Step 2B Preannealing Ampli WGA Kit Ampli1 Whole Genome Amplification Procedure 2A 3 Incubate all samples accordi
12. on 9 Sample Specification and that the working area is properly equipped Step 1 Cell Lysis 1 1 Prepare Lysis Reaction Mix according to the protocol in Table 1 1 AN It is recommended to prepare a Lysis Reaction Mix for at least 10 reactions even if less than 10 samples will be run preparation of a larger amount of Lysis Reaction Mix needs to proportionally increase all other Ampli1TV WGA reaction volumes needed in this step Tab 1 1 Preparation of Lysis Reaction Mix Volume per 10 Vial Label Cap Color reactions p RI Ampli1 white 2 0 Reaction Buffer 1 R2 Ampli Reagent 2 blue 1 3 R3 Ampli1 Reagent 3 blue 1 3 E1 Ampli1 Enzyme 1 blue 2 6 H2O Ampiii Water colorless 12 8 20 0 per reaction 2 0 Once the Lysis Reaction Mix has been prepared briefly vortex and spin it 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 A Pipette 2 ul of Lysis Reaction Mix delivering it onto the wall of the tube above the other liquid already present 1 ul but without touching it Final volume at this point 3 pl 13 Incubate all samples according to Table 1 2 A Briefly spin down all the sample tubes prior to placing them in the thermal cycler Ampli1 WGA Kit Version 01 page 9 of 16 Step 2A DNA Digestion Ampli WGA Kit Ampli1 Whole Genome Amplification Procedure Tab 1 2 Thermal incubation profile
13. t siliconbiosystems com Telephone number 39 051 40 71 300 Ampli1 WGA Kit Version 01 page 4 of 16 Additional Required Materials 6 Additional Required Materials Thermal Cycler 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 20 C Storage Freezer Vortex 7 Ampli1 WGA Kit Description The Ampli1 Whole Genome Amplification kit has been specifically devel oped 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 a Ampli1 WGA procedure is a library of highly concentrated DNA which can be employed for further targeted genetic research analysis The main features are of the Amplit Whole Genome Amplification kit are Ampli WGA Kit Comprehensive and homogenous amplification of the whole genome isolated from a single cell Robust and reproducible reaction results No need for 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 Sequence complexities of multiple primer binding sites are avoided resulting in optimal amplification conditions for all adapter ligated sequences used Version
14. tex and spin it down in order to collect al the reaction mix A Do not add Preannealing Reaction Mix to the samples Version 01 page 11 of 16 Ampli1 Whole Genome Amplification Procedure 2B 2 Incubate the preannealing reaction mix 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 CC Hold Volume pl 1 65 1 minutes 1 1 minutes samples x 3 pl 15 1 minutes 1 15 oo 9 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 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 Vial Label Cap Color E SH E Sieg Preannealing 3 0 30 0 reaction R6 Ampli1 Reagent 6 green 1 0 10 0 E3 Amplil Enzyme 3 green 1 0 10 0 per reaction 5 0 50 0 3 2 Add 5 ul of Ligation Reaction Mix to each sample A Pipette 5 pl of Ligation Reaction Mix delivering 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 pl A Briefly spin down the samples tube and put them back in a microtube rack Ampli1 WGA Kit Version 01 page 12 of 16 Step 4 Primary PCR Ampli WGA Kit Ampli1 Whole Genome Amplification Procedure 3
15. the fluid directly onto the wall of the tube without disturbing the fluid Always collect all the liquid by a short centrifuge spin after adding reaction mix and before putting the samples in the thermal cycler Version 01 page 7 of 16 Ampli1 Whole Genome Amplification Procedure 11 Ampli1 Whole Genome Amplification Procedure 1 Ampli1 WGA procedure overview All the reactions required for Amplit 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 Add Lysis Reaction Mix to each sample and incubate at 42 C over night Day 1 Step 2 A Add Digestion Reaction Mix to the same tube and incu bate at 37 C for 3 hours Step2 B Prepare Ligation Reaction Mix by pre incubating reagents and incubate Day 2 x Step 3 Add ligation Reaction mix and incubate at 15 C over night Step 4 Add Primary PCR Reaction Mix and incubate with ampli fication cycles Day 3 UC S Wh Fig 1 Amplii Whole Genome Amplification Procedure Ampli1 WGA Kit Version 01 page 8 of 16 Ampli1 Whole Genome Amplification Procedure 2 Ampli WGA procedure and reaction Before starting make sure that all the samples meet the requirements described in secti
16. uct 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 01 page 15 of 16 Appendix A 14 Appendix A An example of The library of fragments generated through the Ampii1 WGA procedures use of Ampli1 originates from DNA digested as follows WGA outputin e TYTAA 3 PCR downstream 3 AATAT 5 research assays The specific action of the Digestion Enzyme make it possible to determine the exact sequence of Amp 1 amplification products around any target region Primer design Designing target specific PCR to amplify and analyze one target sequence of an the Amp i1 WGA amplification product requires specific co
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