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Bacterial conjugation


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#1 Alex333

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Posted 17 February 2009 - 04:23 AM

Hello,

I'm currently trying to conjugate the E. coli strain SM10 with a Salmonella Typhimurium strain.

The protocol is as follows:

- I grow overnight cultures of the two strains in LB separately
- the next day I plate 200ul of each culture on one LB plate
- I incubate overnight at 37C
- the next day I collect the bacterial smear in some mls LB
- I make dilutions (1/2, 1/4, 1/8, 1/10) in HBSS and plate 300 ul of each dilution on MacConkey with ampicillin50 (selecting for the Salmonella strain) and nalidixic acid 20 (selecting for the plasmid transferred from the E. coli and integrated in the Salmonella genome)
- I incubate overnight at 37C

The next day I see some large Salmonella colonies on each plate, but also a smear of much smaller salmonellae between the larger colonies.

First I thought that no conjuagtion occured between my two strains, but after testing the obtained large colonies, these clearly were Salmonella Typhimurium that had received the plasmid and had it integrated in their genome.

Now I think that my conjugation occurs rather inefficient because I need some hundreds of separate colonies per plate and thus no smear.


Any tips and tricks for my conjugation?

Thanks in advance!

#2 HomeBrew

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Posted 17 February 2009 - 06:56 AM

The first thing I would try is mating the strains much earlier. Transfer a few drops of your overnight recipient and donor strains to tubes of fresh media, grow these for several hours (until the subcultures are just visibly turbid when swirled). Plate these as before, and see if your efficiency improves.

BTW, are you growing your donor strain with selection for the conjugal plasmid?




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