Site-Directed Mutagenesis Kit; any modified protocols for plasmids larger than 1 - (Jul/28/2011 )
Hey,
Just wondering whether anyone knew of a way to optimize mutagenesis of plasmids of greater than 15kb using the QuikChange Lightning Site-Directed Mutagenesis Kit? The recommendation is for up to 14kb, but the vector I need for the protocol is considerably larger.
Any help or suggestions will be greatly appreciated,
Cheers.
I have some experience in this, but it was some time ago, so i"m going from memory here. The two key changes were to make sure you have adequate elongation time with the polymerase (I used PfuTurbo with good results) and very highly competent cells that work with large constructs. I used XL-10 Gold cells as I found these worked much better than the homemade DH5alpha we typically used. Otherwise, good primer design is always key.
Hope this helps.
Kevin
KevinK on Fri Jul 29 14:28:56 2011 said:
I have some experience in this, but it was some time ago, so i"m going from memory here. The two key changes were to make sure you have adequate elongation time with the polymerase (I used PfuTurbo with good results) and very highly competent cells that work with large constructs. I used XL-10 Gold cells as I found these worked much better than the homemade DH5alpha we typically used. Otherwise, good primer design is always key.
Hope this helps.
Kevin
Thanks a lot for the help, very kind of you. I'll look into those changes, see if it makes a difference. I'll let you know!
A
KevinK on Fri Jul 29 14:28:56 2011 said:
I have some experience in this, but it was some time ago, so i"m going from memory here. The two key changes were to make sure you have adequate elongation time with the polymerase (I used PfuTurbo with good results) and very highly competent cells that work with large constructs. I used XL-10 Gold cells as I found these worked much better than the homemade DH5alpha we typically used. Otherwise, good primer design is always key.
Hope this helps.
Kevin
Also, I just wondered whether you had noticed any particular increase in error rate when using the PfuTurbo over the PfuUltra that is supplied with the kit? I know there's supposed to be a slight increase in rate of errors with the former.
Again, many thanks for your time.
Alex