A question about shotgun sequencing

[Sam0127]

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!

[ecoli]

It is possible... it's difficult (but not impossible) for e. coli to take up multiple plasmid DNA.

[CharonY]

I am not sure what the question is. Do you mean that all E. colis within a well (originating from the same colony) carried the same plasmid with the same inserts? If so, what precisely is the question? Why all clones of the same colony carried the plasmid with the same insert (answer is right there)? Or why a given plasmid only carried one insert? Or why each clone only carried one plasmid with one particular insert?

 

For the last two questions: during ligation you adjust the insert to plasmid ratio so that not more than one insert will statistically insert. For the second, transformation is a relative rare event. Again, the plasmid to cell ratio is generally adjusted to allow only one transformation event per cell.

[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.

[CharonY]

Thanks for letting us know how it worked out.