Direct PCR Lysis Reagent 50ml (tai
Viagen DirectPCR Lysis Reagent is a buffer used to lyse animal tissue and release DNA for future applications, such as PCR. It promises to contain inhibitors of PCR inhibitors found in animal tissues, making the released DNA 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 the simple protocol. Simply add about 200-250ul of reagent and ~ 25ul of 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 are helpful to ensure complete lysis of the tail. The crude lysates are then incubated at 85 ° C for 45 minutes to inactivate proteinase K. For genotyping by PCR, approximately 1 ul of the crude lysate is used. I have used this buffer multiple times. For the most part, it has worked very well. It is much easier and faster to use than DNeasy kits and much easier than creating your own PCR tail lysis buffer.
However, DNA is definitely not as pure and concentrated as when using a kit or making your own buffer with subsequent precipitations and washes. If higher purity is required, it is possible to use Viagen’s DNA lysate and add NaCl and isopropanol to precipitate the DNA, followed by a subsequent wash with 70% EtOH and reconstitution in 10 mM Tris-HCl.
One of the downsides of this system is that it promises to be compatible with only three Taq polymerases (Eppendorf Hostmaster, Sigma JumpStart, and Qiagen HotStar), all 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, and already meticulous PCR reactions have the most problems. Sometimes it is necessary to repeat the PCR using different amounts of DNA, or just start over.
The best feature of this buffer is its price and ease of use. It is very cheap and very fast. Add buffer, proteinase K, incubation overnight and a short incubation at 85 ° C and you have the DNA ready to use in PCR. If for some reason, this does not provide the necessary purity, the DNA can always be purified by simple precipitation with isopropanol or ethanol. However, if it works without it, it is really wonderful. Quick, simple, and inexpensive are three things that cannot be beaten.