B***v
发帖数: 113 | 1 我用QuikChange做mutaion，成功率70%的样子。其余30%没有colony,
该咋办，有经验的大师给指点一下。 |
C*******e
发帖数: 4348 | 2 给的信息太少了，很难帮你trouble shoot啊
引物的设计，质粒的大小，做模版的质粒是否新鲜，一个点还是几个点同时突变.... |
B***v
发帖数: 113 | |
x********e
发帖数: 35261 | 4 直接合成DNA嘛
【在 B***v 的大作中提到】
: 我用QuikChange做mutaion，成功率70%的样子。其余30%没有colony,
: 该咋办，有经验的大师给指点一下。
|
B***v
发帖数: 113 | 5 没钱，有的是时间
【在 x********e 的大作中提到】
: 直接合成DNA嘛
|
M******g
发帖数: 152 | 6 Try NEB Q5® Site-Directed Mutagenesis Kit https://www.neb.com/products
/e0554-q5-site-directed-mutagenesis-kit). It is much much better than the
quick change. They also have a web tool to help you design the primers: http://nebasechanger.neb.com/. Very convenient.
Make sure to use their comp cells. if you use comp cells from other
companies, dilute your final rxn (1:3), then do the transformation.
If your plasmid is big such as 8 kb, I normally double the denaturing time
as NEB suggest.
Another experience using the NEB kit is: do not spread too much
transformation outgrowth, otherwise you will get too many colonies on the
plate. I normally spread 10-20 ul (out of 500 ul outgrowth).
I've used quick change for many years, but after I found NEB kit, I tossed
the quick change.
Hope this helps. Good luck. |
B***v
发帖数: 113 | 7 I'll check it out. Never used it. But sounds so good based on your
experience.
Denaturing time doesn't seem to be an issue as other mutations worked using
the same template.
春节快乐！
products
【在 M******g 的大作中提到】
: Try NEB Q5® Site-Directed Mutagenesis Kit https://www.neb.com/products
: /e0554-q5-site-directed-mutagenesis-kit). It is much much better than the
: quick change. They also have a web tool to help you design the primers: http://nebasechanger.neb.com/. Very convenient.
: Make sure to use their comp cells. if you use comp cells from other
: companies, dilute your final rxn (1:3), then do the transformation.
: If your plasmid is big such as 8 kb, I normally double the denaturing time
: as NEB suggest.
: Another experience using the NEB kit is: do not spread too much
: transformation outgrowth, otherwise you will get too many colonies on the
: plate. I normally spread 10-20 ul (out of 500 ul outgrowth).
|
h**********r
发帖数: 671 | |
g*c
发帖数: 68 | 9 要用好quichchange应该看看下面这篇paper.
Nucleic Acids Res. 2015 Jan;43(2):e12. doi: 10.1093/nar/gku1189. Epub 2014
Nov 15.
New insights into the QuikChange™ process guide the use of Phusion DNA
polymerase for site-directed mutagenesis. |
m******o
发帖数: 8504 | |
B***v
发帖数: 113 | 11 不是还有30% fail嘛
我会试一下上面推荐的Q5
【在 m******o 的大作中提到】
: 成功率70%难道不是很高嘛？
|
m******o
发帖数: 8504 | 12 我们都是直接用Q5 polymerase，不用KIT，就是再普通PCR的基础上稍微调调参数就可
以了。
然后挑5-6个colonies,能搞到1-2个正确的就可以了
【在 B***v 的大作中提到】
: 不是还有30% fail嘛
: 我会试一下上面推荐的Q5
|
B***v
发帖数: 113 | 13 回来汇报一下，3个用QuikChange不成功的mutation，用Q5一举成功，看来QuikChange
可以扔了
多谢宝盖俄州同学指点 |
