Red/ET recombination

Kyle Legate legatek at hotmail.com
Mon Jul 12 14:00:43 EST 2004


Tam wrote:
> Have anyone tried Gene Bridges Red/ET recombination kit?
>
> I got some questions:
>
> 1. If I can't shake my bacteria at 1100 rpm (don't have such a
> powerful shaker), can I grow them longer until desired OD 0.2?  What
> could you recomend? May be aerate them more by growing in a larger
> volume?
> 2. How many times you add dH2O, when you make electrocompetent cells?
>
I don't think I need to know the kit to answer these questions. If you want
to aerate your cells more you have to grow then in a smaller volume, not a
larger one. Or the same volume in a larger flask. The aim is to maximize the
surface area to volume ratio. Using an Erlenmeyer flask with baffles (is
there a specific term for these flasks?) to increase turbulence will also
help. Of course, they will get to 0.2 when they're ready.
When I make electrocompetent cells I wash them 3 times in 10% glycerol (I
wouldn't use water as that may harm the cells), just to wash away salts.


> 3. Have you tried counter-selection kit? Does it work?
>
This I would need the kit to answer.





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