Hello,
I am working on a hybridoma cloning and sequencing experiment. I did RT PCR to produce cDNA followed by amplification on PCR. Then i purified the gel and extracted DNA from the samples. Till this step the experiment is going okay as i am getting a good DNA concentration. Then i am trying to add poly A tail with the help of TOPO TA kit followed by transformation using Multishot stripwell TOP10 cells but i dont see any colonies on the agar plate. Please recommend.
Thanks!
I'd do the A-tailing before you run out samples on a gel. If you get one bright band & minimal or no primer dimers, you can just clean the PCR sample & not use gel extraction.
You can run out a small aliquot of your A-tailed PCR on a gel to check the size of the band, clean the rest of the sample, and use that for your TOPO TA kit.
You can always try increasing the reaction time for the TOPO kit, spreading out more cells per plate, spreading out all of the transformed cells, and transforming more than one tube of cells.
Good luck!
May be you should change the time and/or temperature.
Hi Shreshtha Mody
You can try with a different lot of recipient (competent) cells. Please check your heat shock protocol, make it brief but sharp.
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