Help! PCR Problems
ANDREI.POPOV at afrc.ac.uk
Tue Nov 8 14:49:26 EST 1994
Yang Hsiang Mei (linp.bbs at gopher.ncku.edu.tw) wrote:
: Hi Netters,
: I'm having trouble amplifying an 11kb fragment from total genomic mice
: My PCR result has always been a large smear. I followed the TaKaRa Ex Taq
: kit's protocol with some modifications (temp/time combinations). My
: primers were 30bp & 33bp long. Does anyone have better experiences in doing
: PCR using the total genomic DNA as template, or amplifying large fragments?
: Any help would be greatly appreciated!
>In My books 11Kb isa pretty large fragment. Try using a 2 minute denaturing
>temperature to ensure that your DNA is totally linear. Also try using rtTh
>taq which allows you to increase your annealing temperature to lets say
>60C.This enzyme allows you to have a higher processitivity and the
>temperature may also help you to keep you DNA linear.
>Another important factor is to do a hot start. I think this is VERY
>important in cases where you do a long PCR.
Preheating of large DNA fragments (or usage of long denaturation times in PCR)
for more than 30-60 sec was shown to lead to the marked degradation
of template (Gene, 1993, v.123, 241-244).
Experimentally, it never worked in my hands, while 5-30 sec were
sufficient for amplification of up to 3 kb from genomic template.
I agree that hot-start is a must.
More information about the Methods