Primers and enzymes not compatible - How can I fix this? (Dec/04/2006 )
Hello everyone. I'm trying to make a GFP for injection into C.elegans.
I designed primers using Invitrogen's oligo perfect primer design applet. I know the whole gene + promoter is 7kb, so normal Taq will not cut it. I need to include Pst1 sites at the 5` of both primers, (which Invitrogen's applet does not take into account when guessing the Tm). I retyped the primers into an applet for the enzymes we will be using (either Dynazyme or Phusion) and they are 72-75C!
I have primers that will work under the salt concentrations for a normal Taq, but are way too high for the higher fidelity enzymes we have. I tried shortening the primers, but I lost specificity. Can anyone suggest something please?
I designed primers using Invitrogen's oligo perfect primer design applet. I know the whole gene + promoter is 7kb, so normal Taq will not cut it. I need to include Pst1 sites at the 5` of both primers, (which Invitrogen's applet does not take into account when guessing the Tm). I retyped the primers into an applet for the enzymes we will be using (either Dynazyme or Phusion) and they are 72-75C!
I have primers that will work under the salt concentrations for a normal Taq, but are way too high for the higher fidelity enzymes we have. I tried shortening the primers, but I lost specificity. Can anyone suggest something please?
I do not think you should take the Pst1 sites into account when calculating the Tm. Tm is a meassure of the temperature optimal for hubridisation.
RE sites are not supposed to hybridise to anything the first round of PCR, and if that goes fine - obviously next rounds should also work fine.
You probably need to clone your PCR product into a vector. In that case, specificity is not really an issue as you can gel purify the right product. As long as you can amplify it, you can purify it that way and clone.