We have constructed several plasmids that express HA- tagged proteins. At first, all the protein expression of these plasmids were confirmed by WB. But now, after several rounds of amplification of these plasmids, the expression of HA tagged proteins could not be detected. Does anyone have this problem?
what do you mean by "amplification"? Is this growing the E. coli out from a glycerol stock? Are you always starting from the original glycerol stock? Are you streaking the bacteria out onto a fresh plate first? I suspect that the rounds of amplification are selecting for recombinant plasmids that no longer contain your sequence of interest but still express your antibiotic resistance marker--these clones probably have a growth advantage compared to bacteria containing the full-length plasmids.
I would check the plasmids that you have obtained after several rounds of amplification by a simple restriction digest and gel electrophoresis. If they don't look right, then this is your problem. If so, streak out the bacteria from the original glycerol stock onto fresh plates. Select single colonies and verify that the plasmid is correct by miniprep. Freeze down glycerol stocks at this point and use them for your plasmid midi/maxipreps.
If I misinterpreted your question, I'm sorry.
I agree with what Tai said. You are like get a wrong plasmid after a few rounds of ampilifications. I suggest every time, you need to produce your plasmid from single colony. Or the best way is, to streak out the frozen stock which you know it worked.
There are many examples like this. This is becuase some DNA (genes) are not stable inside E. coli host. It is better to go with fresh transformation before you express your protein. You can just store your right plasmid DNA and make fresh transformation each time. I have got the same impression with one of my protein and I express 19 truncated variants of this protein. All the time I must use a fresh plasmid, otherwise I get nothing. This was the only solution I finally got.
Thanks a lot for your comments. The 'amplification' I mean is that I re-transform the plasmid to E.coli and streak onto a fresh plate. Then I select a right clony by PCR, and the isolated plasmids were also identified by restriction digest. So I think the plasmids were right, but it just don't express protein.
Thanks for all the suggestions. I will try re-transformation again and confirm the plasmids first.
- Badges
- Science topic