Long primers (60bp) and 5kb PCR product problems - (Feb/24/2012 )

I'm trying to add multiple cloning sites to either end of an insert. So my primers are about 30nt MCS and 30nt sequence specific. The GC% is 61 and 51 for the forward and reverse primers. The expected Tm is 74 and 72, respectively. The amplicon will be 5kb long.

I can't get it to work.

I'm using a two step PCR program for my taq:
1. 94 0:30
2. 94 0:30
3. 68 5:20
4. To step 2, 34x
5. 68 7:00

I get a FAINT band and when I try to TOPO clone it and sequence the vector, I see my insert but NOT the MCS that I added.

I'm hoping that a better PCR will result in a better product which will TOPO clone better. I messed up the primer design and forgot to add those buffer bases on the end, so cutting isn't a good option.

Does anyone have any advice on how I can get this to work?
Are my primers too long? Do I need to cut down on the non-specific part of the primers?

Thanks,
Paige

I'd strongly suggest trying an explicit annealing step, something like this for the loop:

94 0:30
55 0:30
68 5:20

I think you are not allowing the primers to anneal, and the 55 for 30 seconds anneals the primers. This would be especially important for the first few cycles, where only the 3' end of your primers are annealing.

Thanks! I'm going to give that a try today.

The Tm is for whole primer or just for the specific part? You should calculate with the Tm of specific part only and adjust the annealing accordingly, if 55 doesn't work.

The 55 PCR didn't give me any stronger band than I had before. I did 5 cycles at 55 then 34 cycles with the two-step.

The sequence specific Tm's are 68 and 62. I guess I could try the same PCR at 60 but I'm not confident that it would do better than the 55. Any ideas?

Do all cycles at 55 (or other annealing temperature) not just five, that's not enough. The 68 may be too high for your primers, and they don't bind the template efficiently. If you want to keep high specifity, do it other way, first cycles at higher temperature, then lower the annealing. That's called touchdown. But try 35 cycles at 55 first to see what it does.