I am trying to do a Site directed mutagenesis and after DpN1 digestion,I transformed the PCR product in DH5 alpha cells and subsequently plated it on kanamycin containg agar plate since my plasmid contain kanamycin resistance gene and performed plasmid isolation from the colonies observed the next day.However after sequencing I got colonies carrying a different gene.I had prepared fresh competent cells and cross checked it by plating on kanamycin plates,so there is no contamination in my competent cells.Can anyone tell how can a bacterial colony carrying a entirely different plasmid with the same antibiotic resistance grow on my plate?
It could be from contamination. A few sources to check would be your pipets, your solutions, or maybe your plasmid prep is actually a mixture. Do some control experiments to see if you can find the source of the problem.
I would also make sure (if you haven't already) that it's not just due to a bad sequencing run; are you sequencing using primers based off the plasmid or your gene of interest?
You may need to do PCR troubleshooting. Before Dpn1 digestion when you were doing PCR, may be the extension time is very less or other issues it facing so full length to plasmid is not getting amplified.
You must know that after PCR you're getting a linear DNA, and it's getting ligated into a complete vector after you've done with transformation, and within bacteria, it's free to get recombinated. So chances are many.
I would first recommend you to run AGE after PCR to check whether you're getting the full length of DNA of interest or not. If yes, then you can repeat the procedure with same protocol, this time you can get positive colonies.
Make sure you have kept control for every step.
Aaron P Landry the sequencing was done using primers based off the plasmid.
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