PCR

I finally got the desired product. Yields from 2 mM and 1.75 mM reactions were about equal. I used 50ng of genomic DNA in each and 0.5 uM of each primer, with a final volume of 50 ul. I tried one tube with 100ng of genomic DNA but didn't have a very good product. Program was hotstart 25 cycles of [1min at 94, 2min at 50, 3min at 72] with an initial denaturation and final extension. The bands were fairly faint, so I'm going to try increasing the program length to 40 cycles.

I'm going to take all my posts from the period when I was using a bad primer offline. If anyone desires to see them, contact me.

My mistake... Progress redux

Many thanks to Matt Hamilton and Jan-Feng Cheng at Lawrence Berkeley Labs. Matt set up a few new reactions for me with different enzymes and DNA prepped by their labs. Turns out, though... that I complimented the sequence for the reverse primer but forgot to reverse it! New primer is on its way.

Before this point, I'd been unable to get any products from my PCR reactions. I removed my detailed posts from this blog, though they are stored offline if anyone cares to see them.