Problem amplifying plasmid - (Oct/15/2009 )
I am having a really difficult time with troubleshooting a PCR reaction thats not working. I am not seeing any PCR bands. I have narrowed it down to the problem either being because of the primers or the plasmid template. Is it possible for thereto be something wrong with my template. I checked it on a gel, there's a band at the correct place i.e. the miniprep to get the template was successful.
what about digesting the plasmid (outside of the amplified region of course) to prevent it from supercoiling? Too little and too much of template can inhibit the reaction as well