pcr trouble!!! heeeeeellllpppppp!! - (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.
Step 1: retry the PCR using 10x and 100x diluted template plasmid
Step 2: redesign your primers, perhaps with some help. Primer3 is a good program, and the IDT primer analyzer is good (idtdna.com)