Is necessary to use pfu polymearse for PCR amplification of pre-miRNA for clonin
Posted 07 September 2009 - 07:46 PM
I wonder if I'm necessary to use pfu polymerase for PCR of pre-miRNA.
I think that several taq polymerases have no proof reading activity. So, pre-miRNA which i want to clone may have incorrect sequence.
It have impact to secondary structure or pre-miRNA or miRNA.
Anybody have suggesion for me.
Thank for help
Posted 08 September 2009 - 12:26 AM
pfu is ok. But if your fragment is less than 1kb, you can use taq. Pick several clonings for sequencing (usually 3-5), then chose the right one.
In our lab, we use taq:pfu=30:1 (Sorry, I'm not sure. You can try).
Posted 08 September 2009 - 05:33 AM
Posted 08 September 2009 - 07:13 PM
Posted 08 September 2009 - 11:10 PM
I decide to use taq pol. of BIOTOOL
and select cloning colony around 5 for sequence and select the righ one for transfect.