Proof-reading and Taq mix - (Jun/17/2008 )
Is it possible to use a mix containing a proof-reading enzyme and a normal Taq?
The thing is I am only able to amplify this one gene with Taq (it doesn't work with Vent or Phusion). I've been trying to optimize the conditions but it's not working (at all) and these enzymes are quite expensive, so I'd like to use as less as I could.
Can I do an amplification using a mix of the enzymes to guarantee that I get amplification with Taq and that the proof-reading still does its job?
Maybe it's asking too much, but it's worth asking if someone has ever tried this...
I don't see a problem with that. I think that's what those mixes usually are any way. It's strange that you can get it with Taq but not Phusion. You can always just clone with Taq, it's not the end of the world that this is the only enzyme working. Most of the inserts should be ok. It just means you might have to sequence two or three clones if the first one you try has a sequence change.
I have done 9:1 Taq:Pfu, and I have seen protocols with up to 50:1.
I'll try the combined mix. If it doesn't work I'll just send several clones to sequencing.
Like you said: It's not the end of the world