I have cloned my 3.3Kb insert having repetitive sequence in yeast expression vector, by directional cloning in E.coli. Positive Clone of 8.7Kb was confirmed by restriction analysis. While the same positive E.coli transformants when grown in 100ml LB, the restriction pattern observed is different. But the the digestion pattern is as expected when it is isolated from 5ml culture. Could anyone please let me know why this discrepancy observed from 5ml to 100ml scale up.
I am suspecting you don't have a clone. The digestion pattern that you are seeing is actually nicked, linear and supercoiled forms of plasmid that appear in the gel. The scale to 100ml might be giving you different levels of these plasmid species. My suggestion is sequence the clone. Try digestion with one enzyme to linearise the clone.
The more doublings of your population, the higher the probability that mutants bearing deletions will arise and overtake the culture. Most likely you already have mutants in the 5 mL culture, but at proportions low enough to pass below the limit of detection of your assay (restriction digestion + gel electrophoresis).
Have you tried E. coli strains designed to propagate repetitive sequences, such as SURE or STBL3?
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