发信人: easylifee (消息), 信区: Biology
标 题: Re: question about PCR with very small amounts of templates (<1
发信站: BBS 未名空间站 (Fri Aug 26 16:01:52 2005)

Try to increase the time for annealing step (55C), to 30 to 60 sec may be?
When very small amount of template is using, it take longer for primers to
find their target.
I believe all the smear and smaller bands are non-specific amplification due
to the exra cycles.

Good Luck.


【 在 calliper (calliper) 的大作中提到: 】
: I am trying to get PCR products from very diluted PCR template (<1 ng)
: I used the cycles conditions below to amplify PCR 1 kb from 10^-6,10^-3, 1,
: and 10 ng PCR template. I only got nice sharp band for 10 ng, but smear and
: multiple bands for 1 ng, and smaller bands for 10^-6 and 10^-3.
: Any suggestion to improve it so that i am able to get PCR with small
templates
: ?
: Thanks a lot
: Cycles
: pfu polymerase
: 95C for 3 min
: ...................



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