Single Cell Whole Genome Amplification Kit

 

The Single Cell WGA Kit efficiently amplifies total DNA from single cells or their DNA equivalent about 1 million-fold to produce 2–5 micrograms of amplified DNA in under 3 hours. The kit can also be used successfully in situations where the low input amount is uncertain as it works well over a range of inputs up to thousands of cells or several ng DNA to achieve this final yield of amplified DNA. Both AT- and GC-rich regions are represented reproducibly using this kit. With single blastomere and polar bodies, the kit is able to achieve 95% amplification success rate. The kit performs reproducibly and generates greater than 90% correlation coefficients for qPCR Ct data from replicate single-cell reactions. This method features single-copy sensitivity as well as high specificity with an expected 5 PCR cycle delay between experimental samples and non-template negative controls. The Single Cell WGA Kit produces amplified DNA fragments suitable for Copy Number Variation (CNV) analysis using oligonucleotide aCGH or qPCR; SNP genotyping, mutation detection and sequencing.

 

Image 1: Workflow


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article# description amount net list price
E2620 S Single Cell Whole Genome Amplification Kit 12 rxn 350,- €*
E2620 L Single Cell Whole Genome Amplification Kit 50 rxn 1250,- €*

*NEB recommends high quality PBS for optimal results 20x PBS from Cell Signaling Technology (#9808)
- now free of charge with each Single Cell WGA Kit*

 

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Image 2: 

Example of Background Subtracted RFU amplification curves for replicate single-cell and control no cell WGA reactions that were monitored on a Bio-Rad I Cycler iQ.





 

 

 

 

 

 



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Image 3: Gel image of reaction products.

The size range of amplified product from a sorted single cell or 15 pg DNA (diluted human genomic DNA equivalent to two diploid human cells). 10 μl of 75 μl reactions were analyzed by 1.4% agarose gel electrophoresis: Lane 1: no cell/DNA negative control, Lanes 2 and 3: independent reactions using 15 pg human genomic DNA. Note: The appearance of amplified product will be identical whether the starting material is a single cell or this amount of DNA. Marker M is 2-Log DNA Ladder



 

 

 

 

 

 

 

 


Required Materials Not Included:

  • PCR thermal cycler (Recommended: Real-time qPCR instrument) 
  • PCR tubes or 96-well plates for cell extraction 
  • PCR plate seals for pre-amp steps (Recommended: Microseal® 'F' Foil film, Bio-Rad Cat. #MSF-1001 or Axymat silicone sealing mats, Axygen Cat. #AM-96-PCR-RD) 
  • PCR optically clear plate sealing film for amplification steps (Recommended: Microseal "B" film, Bio-Rad Cat. #MSB-1001) 
  • Low Binding Barrier Filter pipette tips 

Note: Considerable (> 5 μl) evaporation may occur during step 6 of the Pre-Amplification Protocol if the incubation is being performed in a PCR tube or plate without a tight seal. Evaporation may reduce the robustness and reproducibility of the Single Cell WGA Kit. A mock step 6 incubation from the Pre-Amplification Protocol using 15 μl of water is recommended to confirm the suitability of the selected tube or plate/seal combination. 
PBS buffer for cell washes (Recommended: 20X PBS, Cell Signaling Technology Inc. Cat. #9808 or 10X PBS, USB Corporation Cat. #75889) 

  • DNA purification system (Recommended: DNA Clean & Concentrator™-5 Kit, Zymo Research Cat. #D4014 or MultiScreen PCR96 Plate, Millipore Cat. #MSNU03050 or QIAquick PCR Purification Kit, Qiagen #28104) 
  • SYBR Green I dye (Recommended: Invitrogen Cat. #S7563) 

Note: Dye can be diluted to an intermediate concentration in DMSO for storage at -20°C. For the final working stock, this intermediate stock can be further diluted in 1X TE (pH 8.0) for storage at 4°C for up to 1 month.