发信人: royluo (roy), 信区: Biology
标 题: Re: who has experience with PCR-based mutangenesis?
发信站: The unknown SPACE (Wed Sep 11 14:04:46 2002), 站内信件

1 design primers of ~30 bp length.
2.Use good polymerase to do PCR (herculase from stratagene is great,
a Pfu with special buffer in it to increase the fidelity)
3. add 1~3 ul Dpn I directly to the PCR reaction tube. digest it
for at least 3 hours in 37 degree
4.Desalt the PCR sample with PCR purification kit from Qiagen.
5.Transform competent E.Coli.
6. Pick up several colonies , grow small cultures and do miniprep.
7. Send the DNA to sequencing.

【 在 golem (狗愣小岩) 的大作中提到: 】
: that one developed by Stratagene using DpnI to remove original plasmids.
: I plan to do some without buying the kit. If anybody has experience, please
: give me a list of critical steps and useful tips.
: thanks, and kow tow.


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