Viagen DirectPCR Lysis Reagent is a buffer used to lyse animal tissue and release the DNA for future applications, such as PCR. It promises to contain inhibitors of the PCR inhibitors that are found in animal tissues, thus making the DNA released compatible for subsequent PCR genotyping. There are a few different buffer composition options available for purchase: tail, ear, yolk sac and cultured cells. The best thing about this buffer is the ease of use and simple protocol. You simply add around 200-250 ul of reagent and ~25 ul proteinase K (20 mg/ml) to the tail sample. The tube is incubated at 55°C for 4-6 hours, intermittent mixing and vortexing of the sample is helpful to ensure complete tail lysis. The crude lysates are then incubated at 85°C for 45 minutes to inactivate the proteinase K. For PCR genotyping, approximately 1 ul of the crude lysate is used .
I have used this buffer several times. For the most part, it has worked very well. It is so much easier and faster to use than the DNeasy kits and much easier than making up your own PCR tail lysis buffer. However, the DNA is definitely not as pure and concentrated as when using a kit or making your own buffer with subsequent precipitations and washes. If greater purity is needed, it is possible to use the Viagen DNA lysate and add NaCl and isopropanol to precipitate out the DNA, followed by a subsequent 70% EtOH wash and reconstitution in 10 mM Tris-HCl.
One of the drawbacks of this system is that it promises to only be compatible with three Taq polymerases (Eppendorf Hotmaster, Sigma JumpStart and Qiagen HotStar), all of which are more expensive than some other brands. I have noticed that this DNA also works well with our Qiagen Taq Polymerase and not the cheaper Promega Taq – I have not tried the other Taq polymerases. It also seems to work better with some protocols than others, with already finicky PCR reactions having the most problems. Sometimes it is necessary to repeat the PCR using different amounts of DNA, or simply start over again.
The best feature of this buffer is its price and the ease of use. It is very inexpensive and very quick. Addition of buffer, proteinase K, overnight incubation and short 85°C incubation and you have DNA ready to use for PCR. If, for some reason, this does not give the purity needed, the DNA can always be purified by simple isopropanol or ethanol precipitation. If it works without, though, it is truly a wonderful thing. Fast, simple and inexpensive are three things that cannot be beat.
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Comments? Questions? Discuss this review of DirectPCR-Lysis-Reagent-From-Viagen with the author of this review, Elizabeth Hawkes, and the rest of the Biocompare community.
on Jul 25, 2008 ETk66 said:
I'm kind of new at this. So is this like the Ezway Direct PCR buffer?
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Elizabeth Hawkes
Research Associate
Department of Ophthalmology
University of California, San Francisco
United States
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