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.
- For the size of your adult PCR control bands I would say yes reduce your % agarose to 0.7%
- However that doesn't explain your embryonic cDNA samples with null bands
- You have validated your primers but presumably not the embryo gDNA preps
- You could simply try diluting your samples 1:2 to 1:5 and repeating PCR with 1uk of your lysate prep. I have known this work for genotyping PCR
- Specifically what you actually hope for to successfully PCR from genomic DNA is 10ng to 100ng with 20ng to 50ng being optimal for most primer/prep combinations ( it does depend on those 2 parameters). Ordinarily from ear snips or tail chips this ~50ng will be present in the 1ul you routinely use
- However In your situation you could nanodrop and make sure that your concentration from 260nm reading approximates to 50ng/ul; as I'm assuming it will for validated adult preps
- Alternatively When PCR genotyping using an identical method to yours with refractory samples I will
Thanks for your answer. I did PCR using primers for another gene and it worked well, meaning that DNA quality shouldn't be the main cause. I also reduced the gel to 0.8% and it did not result in a significant difference. Now I am suspecting that there might be something going on at the locus I was trying to amplify (e.g. compact chromatin structure) and my crude DNA prep was not able to linearize that part of DNA rendering PCR difficult. I am going to use a kit as well as the method you suggested to prepare DNA and hopefully get some products.
Thanks Hualin. I neutralized NaOH with Tris/HCl and incubated with proteinase K. I also used less amount of DNA for PCR and am now starting to get bands!
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