发信人: glider (大苹果), 信区: Biology
标 题: Re: question about PCR急坏了
发信站: Unknown Space - 未名空间 (Thu Apr 1 22:32:19 2004), 站内信件

another suggestion:
double your primer quality - there are some design principle to avoid certein
designs are more prone to error

are you using one step or two step? I perfer two steps RT
a pair of good primer sometimes is the key

--- in our lab, once there were 3 attempts to clone a gene, only
works after finally combine two primers from 2 different attempts
....



【 在 Hangout (山人) 的大作中提到: 】
: 1. reduce your template amount (50 ng or less)
: 2. try touchdown as suggested by other
: 3. or reduce annealing temperature to 50-55 if your primers have Tm 60 oC
: 4. if still does't work, try other Taq polymerase such as roche's
: good luck
: 【 在 beebee (^_^) 的大作中提到: 】
: : 有人做过约1.7kb 的cDNA 扩增吗？
: : 我试了很多次，时而有smear，时而没smear ,就是没有我要的band.
: : 我还试着延长了extension time到2分钟，b/c the fragment is big,
: : also 调整MgCl2多次，没用，顶多就能看到smear.
: : actin 的control是好的,.说明我的cDNA 是好的。
: : 怎么办？老板看偶不顺眼了。


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