发信人: ASATHOR (霹雳火), 信区: Biology
标 题: Re: 撞鬼了.大侠们帮我看看
发信站: BBS 未名空间站 (Mon Aug 15 01:03:44 2005)

From my experience, I think the mutationa are likely created during PCR,
assuming none of the mutations locates within primer region.

I have never used vent before(I used pwo, pfu, and etc). But keep in mind that

even high fidelity DNA pol has its limits(I had PCR-cloned a 3kb fragment
using pwo,picked one colony, found 2 mutations). Therefore, it is not a bad
idea to try something different,eg. another DNA pol with higher fidelity.

It could also be possible that the PCR conditons are somehow sub-optimal,
which could greatly reduced enzyme fidelity.

Another thing you can try(may sound a little stupid)is try to replace the
mutation by a second step cloning. That is, if colony N has a mutation at
position n and colony M's at m, and if you can find a unique restriction site
inbetween m and n (or better, an enzyme can cut twice, one inbetween,one
outside), then maybe you can use same enzyme(single or double digestion) to
cut both M and N and use one as vector another as an insert.
Although, this is totally opportunistic.


【 在 NotThree (大道悠游) 的大作中提到: 】
: 1.)我一直用vent, 觉得还是比较不错的.
: 2.)一开始发现问题,我就继续挑克隆测序, 但是发现所有跳出来的克隆都有突变,而且
突
: 变各不相同.
: 所以觉得再挑下去,也没多大戏
: 3.)模板肯定没问题, 从stock提出来的质粒,测过序的,信号好得很. 而且用同一个管子
的
: 模板已经构建了8个constructs, 没有出现过任何问题.
: 所有都是跟以前一样, 唯一不一样的是引物上面加的不同的的tag(引物的配对部分都是
一
: 样的).
: 郁闷啊...



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