Question about Quikchange kit - Do I really need to buy the kit? (Mar/18/2006 )
Hi guys. I was looking at the manual for QuikChange Site-directed mutagenesis kit from Stratagene, and am thinking that I don't even need to buy the kit to do mutagenesis.
Can't I just buy some DpnI, some polymerase and use some home-made DH5alpha instead of the Xl1-blue supplied by the kit? It seems like I can do the same procedure for a lot less money.
Just to make sure, I'd like to ask: Is it OK for me to use DH5alpha instead of XL1-blue to transformation after cutting with DpnI?
Yes, you can do this. You need to make sure the PCR enzyme you use is not strand-displacing. Pfu, Phusion, Vent all are in this category. Pfu turbo (or ultra) come with the kit. Any Taq or Taq containing mixture (most of the "high fidelity" mixes included) will not work.
DH5a should work fine for transformation.
Also, remember that the template has to come from a dam+ strain to allow DpnI to cut it.
Hi thanks for the reply. I will try using PfuUltra. However, I'm just wondering what "strand displacing" means?? Why does PfuUltra not have this activity while Taq does?
Taq is a strand displacing enzyme. This means that when it encounters an already bound primer on the template strand, it kicks the bound primer out of the way and keeps going. If this occurs in the Quikchange process, then the mutanting primer is displaced by Taq after it completes a circuit of the target plasmid. It then duplicates the unmutated sequence on the template DNA, eliminating the desirable mutation.
I doubt very few people who use the quikchange method actually purchase the quikchange kit.
Most people know better.
Just my thought,
Matt
DH5a cells cannot take the place of xl1blue cells, bcoz there are two nicks on the plasmid after quikchange. Xl1blue cells can fix these nicks but dh5a cannot.
You can use the DH5a strain - we're using it an all our Quikchange experiments. Works fine.
You can use the DH5a strain - we're using it an all our Quikchange experiments. Works fine.
Same here, I can get mutant clones by using DH5a for transformation.