I am capturing B cells from Lung tissue using LCM, and then isolating the DNA for various downstream assays. When I finish DNA isolation using the Qiamp mini kit, I am getting good quantity and quality of DNA. One thing I am doing is running a PCR on one of our known primer sets with my DNA samples. the primer set is around 500 bp and has not exhibited any bands after several attempts. I also ran a PCR on a primer set which is about 100 bp and this resulted in appropriate bands. Could this be because the DNA was fragmented during some part of the LCM process? Please let me know if anyone can help. Thanks!
LCM generates quite a bit of heat. And I'm assuming that you are using FFPE or frozen sections? If it's the former, the fixation process automatically causes issues and breakage of the DNA/RNA.
Usually... LCM for DNA/RNA we usually detect short sequences.
Shen-An Hwang yes, the sections are frozen lung tissue from OCT embedding. I'm also using IR laser and not UV.
Did you add the OCT directly into the trachae to inflate the lungs? The reason was we did this a while back, and the penetration of the OCT was not that great if you just drop the lung section in there, so there was a good chance of cell death.
Anyway, we didn't pursue it very far.
To see if your DNA is fragemented, you can just run your samples on an agarose gel and see what the bands look like.
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