| | Re: Odd Gel Run following PCR
I'm not sure which well will have the size I'm looking for. The DNA I amplified is a polymorphic region with a 48bp VNTR. The 4R allele should be around 611 bp.
I performed another reaction with poor results as well. I made some modifications though.
) I dropped down to 40 cycles from 44
2) I ran a PCR Temp Gradient from 58-63 C
3) I took out the 55 C step.
I'm unsure about the giant black spot on my PCR. I any event, lanes 3-11 are my temperature gradient from 58-63. Lane 5 is my control. Lanes 3, 7, 9, 11 are control 1, and Lanes 4, 6, 8, and 10 are another control2 . There seems to be a consistent band for Control 1, although you can sometimes see it in Control 2. As always the bottom bands are indicative of rampant primer dimerization which I haven't figured out yet. There at least seems to be bands from non-specific binding as indicative from my previous post, although I'm not sure either if this one band I'm seeing is non-specific too. One of the bands I'm looking for is ~611, although other VNTR polymorphisms might be present in 48 bp intervals. Pretty lost as to what my next run should be. Perhaps higher annealing temperatures?