Unexpected antibiotics resistance - (Jan/18/2006 )
Can somebody help me? i've transformed Ecoli DH5alfa with a ppicZalfaA plasmid, wich carries the sh ble gene confering resistance to zeocin. But I plated in LB+Ampicillin and 24 hours, some colonies grew up, then i have my recombinant plasmid selectioned by ampicillin, not by zeocin. My original strain(dh5alfa) didn't carry any resistance. Can anyone explain me that? anyone has read about that before?
Thanks
how big were the colonies?
are you sure your amp is good? I am thinking 24 hours is a long incubation time for amp at 37C; they may be satellites or colonies that otherwise arose as the amp degraded
Amikins, there has to be origional colonies before there are satellite colones. They grow around the amp resistant colonies (I know you knew that ).
While I have not worked with the plamid your write about, I am pretty sure Zeocin is used for eukaryotic expression, not prokaryotic.
Look at your plasmid map, if it carries two genes, one for zeocin resistance and one for amp resistance then you might have recombinants.
Did you plate a negative control? How many colonies were on that.... or have I misunderstood your entire post??
Hi,
This is possibly just me being paranoid, but when you say that your DH5a didn't carry any resistance, is this something that you have checked for this particular batch of competent cells...? We had a problem once in our lab when our stock of competent cells was contaminated with a plasmid giving resistance to whatever-it-was (possibly Amp, I don't remember). So you should plate some of your competent cells on Amp-plates just to be absolutely sure.
By the way, I can't find the vector you have used. Is it a standard, commercially available vector?
Regards,
Sonja
This is possibly just me being paranoid, but when you say that your DH5a didn't carry any resistance, is this something that you have checked for this particular batch of competent cells...? We had a problem once in our lab when our stock of competent cells was contaminated with a plasmid giving resistance to whatever-it-was (possibly Amp, I don't remember). So you should plate some of your competent cells on Amp-plates just to be absolutely sure.
By the way, I can't find the vector you have used. Is it a standard, commercially available vector?
Regards,
Sonja
Thanks everybody for the answers. My strain was ampicillin sensitive, I plated the same competent cell in a LB+Amp and no colonies grew. The vector I used is a commercial vector by Invitrogen, you only have to search in invitrogen website for ppiczalfa or ppicz and go to vector, then you can find the information. Thanks.