Ferland Louis H.
ferlandl at ERE.UMontreal.CA
Wed Feb 14 03:41:29 EST 1996
On 13 Feb 1996, Anna Blom wrote:
> Date: 13 Feb 1996 16:28:53 GMT
> From: Anna Blom <Anna.Blom at bio.embnet.se>
> To: methods at net.bio.net
> Subject: competent bacteria
> Does anyone has a good method of preparing competent bacteria that can be
> My bacteria are OK as long as I do not freeze them (5x107cfu/ug). If I
> freeze them they become almost useless - 105 cfu/ug. I use the standard
> calcium method. I tried to add DMSO or glycerol while freezing bacteria
> in liquid nitrogen, but it did not help. I also tried to grow bacteria in
> low temperature (22=B0C) before treating them with calcium but... I have =
> subclone quite large and difficult construct so I really need to have
> highly competent bacteria. Of course I can buy some from some company but
> they are terribly expensive.
> Any good advises?
I use frozen competent cells only for "easy" subcloning, as they seem to=20
always be *much* less good than freshly prepared cells. So, my first=20
advice is to make fresh cells for any tricky ligation.
The calcium method has given much poorer transformation efficiencies than=
the Hanahan method, in my hands. Also, since it (Hanahan) involves putting=
DMSO in the cell suspension, the cells freeze better.=20
Finally, your problem may be that you freeze your cells too fast (if I=20
understood well, you seem to go straight into liquid nitrogen?). Putting=20
your cells at -20 C overnight in a well padded box, and then at -80 C for=
another day before liquid N2 may do the trick for you.=20
Worth a shot.
Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757 FAX: (514) 843-2719
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