I am using pBR322 as E. coli cloning vector which as we know having marker sites ampicillin and tetracycline, after division of host cells I transferred these colonies to ampicillin- and tetracycline-containing media. Later I screened out viable cells from the media for separation of gene of interest and my desired secondary proteins but while separation of my GOI I found many cells without having my rDNA but viable while I performed screening. I 'm unable to find what might be reason for these kind of false positive results.
First you should stop to use pBR322. This vector is only of historical importance. The empty vector confers resistance to ampicillin and tetracycline. To detect recombinants you may do do replica plating . In many cases you destroy the tetracycline resistance when inserting foreign DNA. Get one of the common cloning vectors with nice multiple cloning site and suitable for blue/white screening (e.g. pBluescript SK, pUC18 ..)
First you should stop to use pBR322. This vector is only of historical importance. The empty vector confers resistance to ampicillin and tetracycline. To detect recombinants you may do do replica plating . In many cases you destroy the tetracycline resistance when inserting foreign DNA. Get one of the common cloning vectors with nice multiple cloning site and suitable for blue/white screening (e.g. pBluescript SK, pUC18 ..)
Hi Deepak,
It sounds like the insert is missing from your pBR322. Try running your recombinant plasmid constructs on an agarose gel to confirm that you aren't using empty pBR322 to transfrom your cells.
Both answers above should be helpfuöl for you. Take another plasmid like pUC18.
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