Problems with a PCR-No bands and I am starting to question my sanity - (Feb/02/2012 )
I am having problems with this PCR, it has worked for someone else in the lab before, so this person just gave me the protocol, concentrations, I did it just like they did and it didn't work. I need to amplify the multiple cloning site of a plasmid in order to be able to sequence my insert.
I changed all the reagents, reconstituted the primers, dNTPs, MgCl2, water, buffer, got positive controls from someone else (purified plasmids)I got a new taq no so long ago. So, I did a PCR totally unrelated to this one and it worked. Yesterday, I repeated this PCR, using betaine and doing a gradient PCR and it still didn't work. I am using the following concentrations per one reaction:
*MilliQ water -25.5 ul
*Betine- 20 ul
* PCR buffer -5ul
* MgCl2 3mM- 3ul
* Primer 1 and 2 0.4 mM- 2ul each
*Polymerase (1.250 U)- 0.5ul
*DNA- 2ul (the concentration should be around 30-70nM)
Final volume: 50 ul
The melting temperature of the primers is 63C and my labmate told me that an annealing temperature of 56 worked for her. So when I did the gradient, it ranged from 46-66.
That seems like way too much betaine. I would cut back to 5 ul and replace remainder with water. You could try different magnesium concentrations. Are you sure your buffer doesn't already have enough magnesium?
Is your template ok?
A couple of observations: