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neuron, I ran the RNA on a gel before treating with DNase, so one thing I can try is running it after treatment. The manual said there would be smears from the salt concentrations, but at least I can make sure something's there.
jerryshelly, I am using the Promega RQ1 kit, and yes it does come with a "Stop" solution which I used according to their protocol.
I am wondering if the Mg concentration in the DNase kit is interfering with the subsequent steps (either cDNA synthesis or PCR)... I treated 0.5 ug in 5 uL, used all of that for cDNA synthesis (20uL reaction), then used 2 uL of that for my PCR. If that's the problem, I can do a phenol-choloform extraction. (protocol also suggests diluting the DNase buffer or using an alternative buffer, i.e. the cDNA or PCR buffers). Any thoughts on if that could be the problem?
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In Topic: Transformation problem
18 February 2013 - 03:27 PM
phage434, on 17 February 2013 - 02:28 PM, said:
Your competent cells are probably not very competent. You should measure, by transforming 10 pg of pUC19 or other similar plasmid. You should get 1000's of colonies if you have good cells. It is a mistake to think that if you can transform cells with 100 ng of vector that it is good for transformation.
See here to make good competent cells (and measure their competence):
http://openwetware.o...competent_cells
See here to make good competent cells (and measure their competence):
http://openwetware.o...competent_cells
Thanks phage434 for your reply! I think my cells are competent, because I tried transfecting with pUC19 and got a lot of colonies! I can also transfect with another plasmid, just not anything with the pENTR/U6 vector. Any other ideas?
