pCDH Lentivector has 500 bp insert - (Feb/19/2010 )
We bought pCDH-CMV-MCS-EF1-Puro vector for constructing stable cell line. First, we try midi prep to amplify plasmid amounts. In the gel for checking purity of plasmid, we found one band about 500 bp although it's only uncut plasmid. We think competent cells are contaminated. So we changed new competent cells. But the result is same.
We sure it's not contaminated DNA. But we can not explain what this band is.
Please help me to solve this problem. Maybe some friends have same problem?
Thank you very much.
How big is the vector? Could the 500 bp band you see be supercoiled plasmid? Try cutting it with a single-cutter and see whether you only get one band.
Did you add the RNAse to the first solution? I've never experienced that before but I suspect this is RNA contamination.
Thank friends. We single cut the plasmid (about 7 kb), but still have 500 bp band.
I don't think RNA contamination because we used same kit for other plasmids. Only pCDH back bone has this insert.
Woow, we have pCDH-CMV-MCS-EF1-copGFP and guess what, we do have the same "500bp" band there. It is very weak, but it is there. We couldn't get rid of it. It might belong to host bacteria, who knows?
I have seen also that "500 bp" band, and guess what... I am not able to clone anything into my vector! I desperately tried to convert my pCDH-EF1-IRES-gfp lentivector into the gateway system by inserting the gateway cassette (ccdB gene with Chloramphenicol resistance plus att sites). I cut my vector with swa I (for blunt ends) and with other different combinations of REs but until now I don't get any colony on Amp+Chloramp LB-agar plates. On Amp-LB plates I get some colonies, but none is the one I am looking for... has somebody have this problem before? can anyone help me? Thanks!