We genotype adult and fetal mice routinely in our lab. PCR always works smoothly for adults, but the same primers would not produce bands consistently for embryos. As shown in the attachment, the upper and lower lanes are embryos of different transgenic lines, but both knocked in at the Osr1 (odd skipped-related 1) locus. The two lanes on the right are positive controls from adults. DNA was prepared using the dirty method: 50 ul 50 mM NaOH at 95 degrees for 30 min. Average embryonic DNA concentration from Nanodrop was ~600-800 ng/ul. I used 1 or 2 ul DNA for 25 ul PCR reactions using GoTaq polymerase and it did not result in much difference.
As someone from this post (https://www.researchgate.net/post/I_need_help_with_a_genotyping_PCR) pointed out, there seemed to be a lot of DNA stuck in the wells on the gel and not being able to migrate through it. Could it be because too much DNA was used for PCR so that it was not able to be successfully amplified? I ran a gel with genomic DNA and it seemed very intact. The adult DNA's concentration was about 300-400 ng/ul and PCR worked fine if using 0.5-2 ul of DNA. Should I decrease the concentration of my gel? Currently I'm using 1.5% TAE gel.
It's really unexplainable why the same primers always work for adult DNA but not for embryonic DNA. I would appreciate any suggestions and discussions on this topic.