smear in degenerate PCR - (Jan/24/2007 )
I'm trying to do a degenerate PCR using codehop primers. I've tried touch-down, but doesn't work. By increasing the Temp from 56C one cycle to 60C 30 cycles I got once a band of the expected size but very faint... I cut it and use it directly from the agarose to reamplify. I managed to get more product but with a smear all along the lane. Would you try to clone purifying. Would you try reamplifying again with different conditions. Other temperatures don't seem to work.
Try the reamplification with a serial dilution of the template (the cut band). Smears often indicate either too much template or too much polymerase.
I also often gets smears when re-amplifying a faint product I've gel extracted. Generally, the more DNA I isolate from the original PCR, the less chance there is of a smear occuring. I have a couple of suggestions. If you are using a high fidelity polymerase such as Phusion perhaps try increasing the number of cycles to 40 or 50. That sounds ridiculous but Phusion is so good it will do that many cycles without introducing errors. Alternatively, perform 5 x (or more) the same PCR and pool the resultant bands to produce a greater amount of final template.
Thank you for the advice. I'm as well suspicious that the smear has to do more with a low amount of template rather than too much. I'll try pooling several reactions and then using serial dilutions of them.