Sam0127

After I talked to my PI, I agree with that it's possibe but the chance is pretty low. When it happens, if we use the M13 primers (universal sequencing primers) to test the clone by clony PCR, we should see multiple bands. It means that there are different sizes of insertions in plasmids in a single clone.

When I did the shotgun sequencing. The DNA fragments were sheared into 400~500bp then ligased into plasmid vector. These ligation were transformed into competent E.coli, followed by colony screening and 2nd enrichment of selected colony in 96 well plate. The results come out that there is only one sequence (insertion) result from one individual well. Although I knew the E. coli in each individual well was replicated from a single colony I picked up, I am wondering why won't be more than one insertions existed in a single E. coli? Is it possible that different insertions were uptaken into the same competent cell in the earlier step? Thanks!