primer does not bind - help (Jul/29/2005 )
Hi, everyone! I have a big problem with my PCR. Any help will be greatly appreciated.
My full length gene is already in pCAGGS and A/T vector and I was trying to subclone one domain of this gene from these two vector. My PCR never worked!! At least I tried 4 different primers. Everything must have been double checked and done properly. The only possibility might be the primer did not bind due to the secondary structure.
Should I linerize the plasmid? Please give some suggestio.
please give some suggestion
If you want help you needed to give us more information on the conditions you are using, size of insert, etc.
Linearizing your plasmid may help as supercoiled plasmid can "snap back" quickly in the PCR reaction and prevent binding of the primers. This is generally becomes more of a problem with large PCR products. Just don't linearize in the middle of your gene