pfx50 and pfuII - (Jun/26/2006 )
I've been trying to amplify a 450pb fragment (which amplifies well with regular Taq) with pfx50 and pfuII in order to get it ready for TOPO directional cloning but I cannot get the right size with pfuII (It always shows a 2kb band) and nothing is showing with pfx50. Has anyone worked with one of these polymerase before that could give me a clue about what's going on.
Thanks
As you amplify with regular taq, you're template and primers are most likely okay. Apart from this: have you tried at different annealing temperatures, different amounts of magnesium? If you believe stratagene and invitrogen: these polymerases are extremely processive, so maybe try to decrease your elongation time?
Try to decrease your annealing temperature a lot, if your band at 450 bp doesn't appear then, change all reagents (buffers, primers, dntp's, use fresh DNA, and run a control reaction with your taq next to it).
Maybe a stupid question, but can't you "just" add restriction sites to your primers and do the cloning into a vector with restriction/ligation? 450 bp isn't too much, so you most likely have a lot of choice to ad unique restriction sites to your primers?
Otherwise: try another proofreader that's been used more often?
Try to decrease your annealing temperature a lot, if your band at 450 bp doesn't appear then, change all reagents (buffers, primers, dntp's, use fresh DNA, and run a control reaction with your taq next to it).
Maybe a stupid question, but can't you "just" add restriction sites to your primers and do the cloning into a vector with restriction/ligation? 450 bp isn't too much, so you most likely have a lot of choice to ad unique restriction sites to your primers?
Otherwise: try another proofreader that's been used more often?
Thanks for your suggestions. I'm in the anneling temperature step right now I've tryed 10 different ones. Mg will be my next step and then I will go for elongation time. But I simple cannot use the restriction site alternative once I'm working with an specific and new protein expression vector from invitrogen which requires blunt fragments and other sequences in the primer in order to have the protein cloned in frame. Thanks for your help.