tomlab at rockvax.rockefeller.edu
Thu Feb 1 11:02:05 EST 1996
Evereh G Robert <erobert at mailhost.tcs.tulane.edu> wrote:
>It is not clear to me what exactly hapens when a E.coli cell takes up a
>piece of DNA during transformation. In particular, does a cell usually
>take up more than one piece of DNA and do these compete for
>maintainence? Or, alternatively, do the cells only take up one piece of
>DNA. I am asking this question because I have been doing some difficult
>clonings in E.coli which will be "shuttled" into Streptomyces lividans TK64.
>I have designed my experiment such that only the correct clones will
>replicate in both E. coli and Streptomyces. My uncertainty lies in
>whether I need a large number of cells to transform if the desired
>constructs constitute a small fraction of the total plasmid population.
>I would be very thankful for any input.
Of course the rarer the event the more cells you have to plate to find the right construct. You are transforming plasmids so it does not seem to me that you should have any problem if two plasmids get into the same cell (what will not happen unless you use a very high DNA to cell ratio, calculated on a molar basis). The worse cenario would be that you would have to isolate plasmid again and do a second level tarnsformation with a mixture already enriched in your construct. I hope this helps.
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