AMPLIFYING LONG FRAGMENTS - problems obtaining 3kb fragment (Apr/10/2003 )
I am currently trying to amplify (and finally clone) a 3kb fragment from cDNA. Smaller fragments from the same DNA works fine, but when attempting to amplify 3 kb I get several discrete bands from 1.5-0.2 kb and a very light smear up to 2.5 kb. I tried to use Taq, Vent and Expand High Fidelity polymerases. I would like to have some advice since I don't have that much experience in this area.
your unspecific bands are probably due to unspecific annealling -increase your tm.
if u can please write your PCR cycles program.
for 3 Kb fragment u need at least 2.5 min alongation every cycle- try to increase it too
How did you do your first strand cDNA synthesis, using oligo dT or random primer? I think the problem may due to the quality of your cDNA. Make sure you carry out your RT reaction long enough to allow synthsize long enough first strand cDNA. For long distance PCR, you can use two-step cycling instead of 3-step.
Have you tried Isis DNA Pol from Qbiogene ? I worked fine on my genomic DNA cloning ....
Before you try anything I would suggest you use repeatmasker on the sequence you are trying to amplify.....and also look for high GC content. 3kb amplification is not hard at all if you:
1) designed primers using primer3
2) are using correct Tm
3) have 3min extension time