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Re: who has experience with PCR-based mutangenesis?
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Re: who has experience with PCR-based mutangenesis?# Biology - 生物学
R*s
1 楼
It's called QuickChange Site-Directed Mutagenesis Kit.
Just seach stratagene website for it.
It's very easy to use. One thing is they usually give little
Pfu turbo compared to the other reagents from the kit. So you'd
better buy some extra Pfu turbo yourself if you are doing a lot of this.
avatar
r*o
2 楼
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.
avatar
R*s
3 楼
It's called QuickChange Site-Directed Mutagenesis Kit.
Just seach stratagene website for it.
It's very easy to use. One thing is they usually give little
Pfu turbo compared to the other reagents from the kit. So you'd
better buy some extra Pfu turbo yourself if you are doing a lot of this.
avatar
r*o
4 楼
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.
avatar
N*1
5 楼
太不公平了，我要一个说法！！！

【在 R*s 的大作中提到】
: It's called QuickChange Site-Directed Mutagenesis Kit.
: Just seach stratagene website for it.
: It's very easy to use. One thing is they usually give little
: Pfu turbo compared to the other reagents from the kit. So you'd
: better buy some extra Pfu turbo yourself if you are doing a lot of this.
avatar
N*1
6 楼
太不公平了，我要一个说法！！！

【在 R*s 的大作中提到】
: It's called QuickChange Site-Directed Mutagenesis Kit.
: Just seach stratagene website for it.
: It's very easy to use. One thing is they usually give little
: Pfu turbo compared to the other reagents from the kit. So you'd
: better buy some extra Pfu turbo yourself if you are doing a lot of this.
avatar
N*6
7 楼
Jon does not have fund...
I am sad...

【在 R*s 的大作中提到】
: It's called QuickChange Site-Directed Mutagenesis Kit.
: Just seach stratagene website for it.
: It's very easy to use. One thing is they usually give little
: Pfu turbo compared to the other reagents from the kit. So you'd
: better buy some extra Pfu turbo yourself if you are doing a lot of this.
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