哪位TX做过 Single site mutant?# Biology - 生物学
s*l
1 楼
OPT马上就要开始了，找工作找的进展也不大，想一边在这边找一边找一下国内的位置
，但是不知道国内的位置应该怎么找，有什么网站比较靠谱一点的网站吗？这里的朋友
如有知道能否告诉一声，万分感谢！
，但是不知道国内的位置应该怎么找，有什么网站比较靠谱一点的网站吗？这里的朋友
如有知道能否告诉一声，万分感谢！
c*r
2 楼
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
谢谢
s*l
3 楼
自己顶一下。
D*g
4 楼
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!
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!
e*7
5 楼
51job
V*f
6 楼
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/
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
7 楼
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!
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
8 楼
自己看kit的说明书，照着做就可以了。不用买kit。
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