promoter cloning PCR problem - what was wrong for my PCR set-up (Jun/29/2010 )
I was cloning the promoter region of one specific plant gene these days by genome walking. After I constructed the genomewalk libraries, I tried to amplify the upstream region of this specific gene by nested PCRs (primary and secondary PCR). However, I always got some sharp smear at the high molecular weight area on my gel, while very weak bands around 1-2 kb long (see the attached gel picture). Did anybody here encounter this kind of problem before like me and do you know where I was wrong during the process of promoter cloning? Thanks for your attention and I am very appreciated with your help in advance.
Your gel shows very large amounts of genomic DNA. I would suggest retrying your PCR reaction with 100x to 1000x times less DNA as template. The good news is that you likely have your desired product in the bands that appeared.
phage434 on Jun 29 2010, 08:41 PM said:
Thanks, phage434. I diluted the primary PCR mix 50 times for secondary PCR. so it seems that even 50 times is not enough...
In the past I also had problem with cloning a promoter.
In my case, what helped me was a different polymerase (the promoter was very GC rich and Herculase II by Stratagene seems to work nicely in this case) and betaine.