I am trying to amplify an infectious cDNA clone of a plus-stranded RNA virus. The clone is full-length genomic sequence of a plus-stranded RNA virus, under the SP6 promoter in a high copy number plasmid with pMA vector backbone. The initial MIDI prep yields were quite good - 6µg/´µl. I recently cloned a 150bp sequence into the original construct and got a yield of 180-200ng/µl in plasmid minipreps. When I tried to increase the yield by doing a MIDI prep, I get very low yields (~80ng/µl). The protocol was repeated twice and with other constructs done at the same time. There was no problem with the other yields. Could insertion of this sequence have such a drastic effect on the replicative nature of the plasmid?
Hi,
Sometimes the addition of sequence might result in toxicity for the bacteria. We had a problem like this in our lab a while ago and the only way to solve it was to insert an intron sequence in the fragment to disrupt it. However, there are other things you can try that might help increase your yield. You need to control the growth of your bacteria and make sure you start the prep before they reach the stationary phase. If you are using a midi kit, you might want to switch to the old fashion method. You can also try a more rich media to grow your bacteria.
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