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gateway destination vector grows on kanamycin plates !!!!!

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#1 Opal



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Posted 13 April 2013 - 06:05 PM


I have a pDest15 plasmid which containes my gene of interest and it has been tested before; so it expresses the N-terminally GST-tagged protein. recently I used this vectore as my template to amplify my gene of interest and clone that gene to a P-entry vector (kanamycin-resistant) so I thought it's convinient because I can use the PCR product directly and the template won't cause any problem it won't grow on the kan plates. But to my surprise I had a ton of colonies all seem to be the first p-entry vector (+my gene) which I had used in my LR reaction in the past Posted Image . one thing that I used to do with my destination vectors was that I never isolated a single colony after transformation of my LR reaction to the bacteria. I used to put the whole transformation reaction into the amp-LB medium and do a miniprep the next day. So now my question is could the kanamycine resistent bacteria (most likely carried over from my LR reaction ) still survive in LB amp. ? Today I tested my Pdest-15 on both amp and kan. plates and I got the equal number of colonies on both !!! Does any one have an experience?

#2 isamila



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Posted 15 April 2013 - 05:16 AM


may be your pDest15 plasmid containing your gene of interest is contaminated. You should isolate a single colony of pDest15 from a glycerol stock, in a selective medium added with ampicillin, and then extract new plasmid from this single colony and sequence it with specific vector and gene primers. If the sequence is OK, you should dilute 10 or 100 times this plasmid to amplify your gene of interest. Afterwards you should perform the LR reaction again and always pick a single colony that you check by PCR before culture;
Good luck, Isabelle

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