发信人: golem (杯杯狗愣~尽吾心与力，与卿共余生), 信区: Biology
标 题: Re: Ligation Problem
发信站: The unknown SPACE (Wed Jun 26 12:49:33 2002) WWW-POST

man, refer to gzip's post.
the ligation is somehow vulnerable.
1. check with your lab fellow to make sure your ligase is still alive.
2. always use latest ligation buffer, you can even smell the thio stuff if you
are using NEB enzyme.
3. critical thing is your insert: never directly use the insert purified from
gel, you need gel purification followed by ethanol precipitation to make sure
the purity. However, you can use gel extracted vector for ligation in small
volume.
4. add enough insert, forget the recommendation by manufacture. I usually use
DNA equivalant to half tube of PCR (50ul system) for one ligation.
5. Use 16 degree overnight, don't rush.
6. Use vector with blue/white screening whenever possible for first step PCR
cloing.
7. Patience and good luck.



【 在 sensor (apple) 的大作中提到: 】
: Hi, folks,
:
: I am being tortured by PCR mutagenesis. My PCR and digestion work fine. But,
: after ligation and transformation, there was no colony at all on the plates.
I
: repeated for 10 times using different restriction sites. Whoever could give
me
: some advice, I will appreciate it very very much!
:
:
:
:

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