Hi, Im making a triple gene construct. In the final cloning step, inserting the third gene in the double gene construct, I seemed to have lost the second gene.. Ofcourse, those enzymes to insert the last gene are different than those for the second gene, so how is this possible?
It is often to see this lose especially if the plasmid contains repeat sequences, such like the same promoter or terminator for different genes. To reduce this recombinant event, you need coli strain, such as NEB Stable. Or you need check a few colonies to make sure you use the right one.
About losing the gene, could it mean not having the derived phenotype? if so, it is very common if there is no selection for the function encoded. In few generations random genetic drift happens there without selection, reproducing many variants without a functional gene; all these while there is a big part or the complete gene with few mutations is still there cloned. Regarding the options of actually losing the whole gene from the construct, I agree with the two previous answers by Ziguo and Jorg, but while it is obvious, check that there is no recognition site inside the gene cloned for the restriction enzyme in the next round.
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