I need to do a pcr on a liquid culture of E.coli. I'm amplifying a 150bp region of gyrA, using primers that I designed. I'm using NEB Next Ultra 2 Q5 MM. For the reaction, I am using 5ul NEB, 0.5 ul forward primer, 0.5ul reverse, 1 ul water and 3ul culture.
This mix worked fine with e.coli grown in LB, but now I am getting no product when the bacteria is grown in different media. I have 9 undefined environmental media that I have done the competitions in, that I need PCR product from. My PCR protocol is: 3min at 95(C), (repeat 24x) 30s at 95, 30s at 67, 30s at 72; then hold for 5min at 72. Any advice on colony PCR technique would be helpful!
I would run pcrs with a sample that works and aliquots of each medium added to it in case there is an inhibitor present in the other media. if so you may have to wash away the medium in saline before running the pcr
I assume you think the problem has something to do with the media you are using? Try spinning the cells down, and do several washes with water. Also, how long are you growing your cultures? If they are overnights, you definitely don't need 3uL for a 10uL reaction. On that note, you can re-suspend your pelleted cells in water and dilute further (also, you can try boiling them to lyse the cells, although you really shouldn't need to). Hope this helps!
I would run pcrs with a sample that works and aliquots of each medium added to it in case there is an inhibitor present in the other media. if so you may have to wash away the medium in saline before running the pcr
Pellet the cells, wash in water and re-suspend in water. Probably something to do with the inhibitors in the media.
3 uL is too much for a 10 uL PCR reaction, which is 30% of the total volume. Reduce it to 0.1 uL - 0.5 uL which will hold sufficient no. of cells and the media components will be diluted 20-100 times to show any inhibition.
using 1ul in the reaction seemed to work for most of the samples for the t24 time point, but not the 0hr time point(?)
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