Hi all,
I am trying to convert the circular single-stranded DNA to double-stranded DNA using PFU DNA polymerase. The ssNA is around 7kbp. The ssDNA has been converted into dsDNA because it is not digested when I digested it with S1 nuclease. However, the problem I face is the PCR product (dsDNA) is in low yield when I run electrophoresis. (The PCR products haven't been purified after PFU amplification). I have tried optimize it using various PCR conditions or used more DNA template, random primer, dNTP, additives, but it was not work.
I couldn't get bight bands. I can get 3 bands or more with bit smear but the bands are paler than the original unamplified DNA. I got high molecular weight smear with pale bands in long annealing and extension conditions. Sometimes with DNA stuck in the well (bright color in the well and smear in the gel).
Is PFU DNA polymerase suitable for amplifying single-stranded DNA to double-stranded DNA? Will I get good yield of dsDNA if I subjected this PCR product into vector and do transformation?
Hope can get your advice and suggestion. Thank you.
qiagen Long Reach pcr kit is capable of longer reads than 5kb but if you want to use PFU I think that shortr sequential amplifications of your target and then ligation may work better. i would not clone the product of a pcr that only works poorly as I think that the quality as well as the quantity will have suffered and you would probably get a good yield of mutated product
Pfu is made to be very high fidelity but it is very slow (about 1 to 2 min for 1kb). So extension time at 75°C should be around 5 to 10min at each step !
It is very likely that the polymerase will fall off the template before the end of the procedure. Maybe would be better to use Phusion instead which is still high fidelity but faster (15-30s/kb).
The point of using a random or specific primer is to select the region you want to amplified. During the PCR, the polymerase by itself is not really involved in this process. As long as you set up the right parameters for random or specific PCR if required...
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