Extremely Low Yield PCR with restriction site addition - Assistance requested for beginning molecular biologist on PCR techniqu (Dec/14/2007 )
I've been trying for some time now to get a pcr reaction optimized for the addition of two non homologous restriction sites into a purified source plasmid template. Ultimately, I will be using this for BAC cloning but my problem is the extremely low yield of my PCR reactions with the relatively appropriate seeming primers (Tm = 58, 11 bp homology) using well tried reagents (taq polymerase hi fi platinum). The PCR worked in silico with serial cloner and I have run my reaction using three distinct Mg2+ concentrations over a temperature range for several different rxn conditions and the following concentrations for primer and and template:
Primer: 0.3uM, 7.5pmol/rxn
Template: ~30.5 ng/rxn (1.2ng/ul * ) and 75ng/rxn
Mg2+ was varied at 0.5-4mM as recomended
and all other values were as recommended by the taq kit.
The only thing I can think of is that somehow my primer concentration is too low. Is it possible that this is the case? I noticed on my gels that I'm not getting even very small bands but I'm not sure if primers would show up.
On some of my reactions I also seem to be getting bands that are larger than the starting plasmid (which I analytically cut and ran out on a gel showing the expected size of 6kb). Although the source plasmid was not derived in house (addgene) I am fairly confident that it is right. If any one can help me figure out what my problem is I would very much appreciate it.
Thanks in advance.
Redesign your primers. If I understand your comment, you have only 11 bp that match your template. This is not enough. You need 18-22 bp of match. You are being fooled by the Tm of the primer given by some program which is looking at the entire primer, rather than the part that binds to your template. This only works for late cycles in the PCR, where most template is previously amplified DNA. Early cycles will only have 11 bp of match, and will have a MUCH lower Tm. I suspect that you will not be able to make these primers work, and you should simply design them again. If you wanted to try them, do a few cycles with an annealing temperature of 40-48 degrees for the first 5-10 cycles then normal cycling. I suspect it won't work, but it might be worth one try. Chalk it up to experience.
Your understanding is correct, but the program does estimate using just the matching region. I ran the PCR again with higher primer concentration and got a significant increase in product band, but still low. The real problem is that the restriction sites I'm engineering in are necessarily g/c rich so I'm worried about hairpinning and self dimerization. I should have included this in my post but my initial attempt with longer forward primer (which is particularly gc rich) resulted in a product whose size suggested "specific" non- specific binding of the reverse primer as a forward primer (since there was a site higher up which looked like an appropriate candidate with only a 2 bp difference on the 5' end). As you may expect, its hard to get a region which is really gc poor in a specific area of dna which is longer than 12 or so base pairs. Still, what you're saying makes sense, and it may be necessary to go back to the drawing board.
Thanks for your advice and I will try redesigning if I can't purify enough DNA from my slightly stronger bands.
i would rather suggest you a very fast and crude method
your problem is low yield so why can't you just elute out the respective band and then redo the pcr with the primers so that there are not many optimal sites for primer binding. I have followed this method for many of my clones
all the best