Dear All, I have received the pLVX-M-puro plasmid in a bacterial stab and I can´t isolate it. I have tried several times, using fresh Amp-plates and Amp-LB media to first grow the single colonies into a 4ml culture and isolate the plasmid using either the Qiagen mini-prep kit or the mini-prep kit from Thermo Fischer. It has been tried by other people in the lab. The end result is a very low DNA concentration (40ng/µl or less, sometimes 0,5ng/µl). I have sequenced part of the plasmid using the CMA and Puro primers with microsynth and I got no sequence. I then send the obtained DNA for full sequencing with plasmidsaurus and I obtained the sequence of the plasmid pLVX-M-puro with only a few mutations, meaning the plasmid is there. The bacteria grow good in the plates and media.
We also went through all the troubleshooting steps from the mini-prep kits and it did not improve the result.
Who did you receive the plasmid from? Perhaps they could send you another stab and/or have suggestions for purification. You could also try carb as it is a more stable analog of amp.
Are any other amp plasmids of a similar size working well for extraction in the lab? One of those could be a great control for the cell growth and plasmid extraction process.
Another though, try transforming a nice lab strain of E. coli with the extracted plasmid. It may be that the cell type received is for a different purpose (say protein expression) instead of plasmid replication.
Heating the elution buffer to 70ºc can increase recovery, especially of large plasmids, by as much as 10 fold.
Thank you for your suggestions. The plasmid was from addgene and we received it twice already. We also tried re-transformation into E. coli from our lab (DH5a), this also didn't work.
The plasmid is small, 9kb, but we also tried to elute with warm elution buffer as Yoav recommended and it also didn't work
Contact customer service from Addgene, it is possible that you were sent the wrong item (unlikely but possible). If other customers are having the same issue it could be a bad batch.
Are any other plasmids working well in your lab for transformation and extraction?
Thank you Amy. I have contacted Addgene, there seem to be no mistakes from their side. We have indeed successfully extracted and transformed other plasmids.
Very odd! Any chance your plasmid needs something particular for replication? Some sort of media addition or temperature?
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