promoter cloning PCR problem
Posted 29 June 2010 - 04:00 AM
Posted 29 June 2010 - 04:41 AM
Posted 29 June 2010 - 05:19 AM
Thanks, phage434. I diluted the primary PCR mix 50 times for secondary PCR. so it seems that even 50 times is not enough...
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.
Posted 29 June 2010 - 07:15 AM
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.