High MW PCR band seen...help needed
Posted 04 February 2010 - 11:54 AM
Using this 2nd set of primers at 60 and 62 degree annealing temperatures, I get a product >8kp! It is the only band. When I lower the Tm I get a smear from 8kb all the way down to 0.5kb. I have tried increasing the Mg2Cl from 1.5 mM to 2 and 3 mM with little to no success. The GC content is about 52% on the target..
I cannot make shorter primers because the 3' end of the target has a 7bp hairpin in it (can't do anything about this) and needs a Tm >59 to get amplification (as seen from colony screening).
Posted 04 February 2010 - 01:39 PM
Posted 04 February 2010 - 04:50 PM
Posted 05 February 2010 - 11:19 AM
Posted 05 February 2010 - 04:02 PM
Posted 08 February 2010 - 01:00 PM
Posted 08 February 2010 - 05:16 PM
Could you do as phage says and run a PCR reaction before cycling it, or just running an aliquot of your template.
How big is your template? Is it plasmid DNA?
Posted 10 February 2010 - 11:19 AM