c*********r
发帖数: 1046 | 1 不用买的KIT， 只是突变一个AA, 自己设计应物。 有什么要注意的吗？
谢谢 | D****g
发帖数: 275 | 2 Design primers F/R to cover you gene, then design primers F1/R1 with the
single site mutation to cover the mutation area. Do PCR1 with F/R1, PCR2
with F1/R, then purify the two products, put them in the third PCR tube as
template, with F/R as the primer, do the third PCR, purify the product, and
then clone into your target vector. Done! | V*f
发帖数: 171 | 3 just design F1/R1 with the mutation sites in both primers and one PCR would
do it, then cut the template with DPN1 and clone into vector, that simple.
You can use this site to design primers:
http://www.bioinformatics.org/primerx/ | c*********r
发帖数: 1046 | 4 my question: the template for F/R1 and F1/R is wt sequence, which will effec
the following PCR(F/R).The purification will reduce the effection?
how many do I need to add the template of third PCR?
thanks.
and
【在 D****g 的大作中提到】
: Design primers F/R to cover you gene, then design primers F1/R1 with the
: single site mutation to cover the mutation area. Do PCR1 with F/R1, PCR2
: with F1/R, then purify the two products, put them in the third PCR tube as
: template, with F/R as the primer, do the third PCR, purify the product, and
: then clone into your target vector. Done!
| h**********r
发帖数: 671 | 5 自己看kit的说明书，照着做就可以了。不用买kit。 |
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