11 March 2020 2 8K Report

Hi everyone,

I am having a few doubts about gateway cloning. We are using gateway cloning for protein expression using pDEST15 as our destination vector. I attached a picture of the pDEST15 map. From my understanding, during the LR reaction...your entry clone containing the cDNA of interest gets integrated into the destination vector (pDEST15) which happens through a process that involves flanking the two attr sites on pDEST15. Thus, this eliminates the positions from attr1 through attr2...removing the gene encoding chloramphenicol resistance and inserting your cDNA there. Therefore, true positive clones would ONLY grow on ampicillin plates. I transformed one of the maxipreps I had on an amp plate and did the same with a chlora plate and they both grew colonies. Not sure why? Shouldn't it not grow on chlora? Sequencing showed that the gene was indeed in the plasmid. I checked the gateway manual and it said (see attached picture 2) that sometimes the CCDB gene gets mutated and becomes chlora-resistant. Does this mean what grew on the chlora plate was one of those mutated CCDB genes that acquired chlora resistance?

Any thoughts are much appreciated.

Kind regards

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