I recently tried to clone a construct (SAT flipper) which confers resistance towards the drug Nourseothricin. I was cloning this gene into a vector [(pGEM-T Easy) which offers blue-white screening] containing short sequences (approx. 500 bp) flanking the target gene in Candida albicans genome. After transformations I incubated the plate for 16 hours at 37 but the colonies were all blue. This is somehow strange to me becuase the beta-galactosidase gene was already not functional since I inserted my flanks along with the gene of interest into this vector. Could this mean that my flanks are no longer inserted in the vector or may be some of the codons in the resistant gene code for beta-galactosidase? Can somebody please tell me what could cause this.
CHeck your controls (vector only ligation control) before concluding anything from here. If you get the same result in that, probably your flank regions are not cloned or during restriction you have made some mistake and lost them. If that is not the case, then go back to your plasmid, confirm the presence of flank region and then proceed for next cloning. It is very unlikely that codons in the resistant gene will code for beta-galactosidase. I dont think that is possible because I have use the SAT flipper and it does not give me such results. You just need to check your flank presence.
Well I double checked my inserts and flanking regions (see attached file) and already prceeding with cloning. I am not sure what could I do to see what's the problem if it persists.
Thanks
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