Help! PCR Problems

Shahram Mori smori at nmsu.edu
Tue Nov 8 02:02:01 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 DNA.
: 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. 
Good Luck.
Cheers,

--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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