AidanRider

I'm sure that the primers work since weak bands do form on the gel, and I don't extract the DNA but rather just directly place the hairs into the PCR tubes. So I can't check the concentrations. And with the low annealing temperature I know the DNA template is AT-rich, about 40% according to a quick GC-content calculator on the web. I figured out recently that the problem is the formation of primer dimers. The primers are almost 30bp long and not the typical 20bp of the others, so I think that's what causing them. I'm extending the annealing time now and am also lowering the extension temperature to 68 degrees instead of the usual 72. Another thing I can probably try is to lower the primer concentration, but it's already at 10 micromolar. I'm also already using MgCl2 to improve specific binding.

I'm using a standard PCR protocol of the Kapa Hifi Hotstart mix that's proven effective with all my previous primers. I'm doing direct PCR with horse hair roots, but now I'm getting very weak and hazy bands when I run a gel. I've played around with the MgCl2 and primer concerntrations, and also used various amounts of template (2 - 7 hairs), but nothing seems to work. When I sequenced the samples with the weak bands the sequencer stopped base-calling close to where the primer ends. I ran a gradient and know that the best amplification temp. is 54 - 56'C, so I think that's alright. I'm increasing the extension time at the moment, and also increased the denaturing temp.