Re; Electroporation

[tfitzwater at gilead.com]

>Subject: Electroporation
>From: David (bartol at bbm1.ucm.es)
>Date: Thu 13 Jul 2000 - 16:08:00 BST
>Hi all,
>we are experienced some problems when preparing e. coli electrocompetent
>cells. May somebody send me a brief protocol for comparing it with the
>mine. I'm specially interested on knowing how many times do you
>concentrate the cells in the final step.
>thanks in advance
>David

Growth  media  makes a substantial difference in electroporation yields and
should  be  investigated  for particular strains of E. coli.  Some cells do
better  if  initially  grown on plates (transferring a colony to the growth
media, while others do better with a 3 ml overnight culture.  Do not use LB
recipes  containing  10  g  NaCl  per  liter  or  arcing  may result during
electroporation.  2xYT media can be used.

Prepare  4x  500  ml aliquots of 2xYT or LB media in 1000 ml baffled flasks
and autoclave.  Remove a 1 ml aliquot for OD blanks.

Inoculate  3  ml  of  media with a few microliters of frozen E. coli cells.
Grow at 37°C for 12-16 hours.
Inoculate  each  aliquot  of 500 ml media with 500 µl of overnight culture.
Remove 300 µl from one flask for an initial OD determination.

Grow  cells  at 37°C while shaking vigorously (250 rpm) to OD600 of 0.5-1.0
(approximately  1  e  10  cells/ml).   Remove 300 µl aliquots approximately
every  hour  for OD determinations.  Graph this data as you get it, so that
an estimation of when to harvest the cells can be made.

Prepare  4  liters  of Type I water by sterile filtration of water directly
from  the  machine.   Do not autoclave.  Do not remove water from a carboy.
After filtration, store at 4°C until needed.

While  growing  cells,  prechill  the  following:   4 liters sterile Type I
water,  100  ml  sterile  10%  glycerol,  the JA10 rotor, 6x 500 ml sterile
centrifuge bottles and 80-100 sterile screw-cap ampoules labeled.

Transfer  the  flasks to ice when the cells reach OD260 = 0.6-0.8, and hold
for  15 minutes.  Transfer the four 500 ml cultures to six sterile ice-cold
500  ml centrifuge bottles (333 ml @).  Spin JA10 rotor at 4°C and 4000 x g
(3700  rpm) for 15 minutes.  Discard the used media.  Keep the cells on ice
at all times from now on.

Chill  the cells on ice.  Using sterile technique, pellet 2 L of culture in
six ice-cold 500 ml centrifuge bottles at 4000 x g at 4°C for 15 minutes.
Wash 1:  Gently resuspend each bottle in 1 media volume sterile cold H2O.
Pellet at 4000 x g at 4°C for 15 minutes.
Wash 2:  Gently resuspend in 0.5 volume sterile cold H2O.  Pellet at 4000 x
g at 4°C for 15 minutes.
Wash 3:  Gently resuspend in 0.5 volume sterile cold H2O.  Pellet at 4000 x
g at 4°C for 15 minutes.
Wash 4:  Gently resuspend in 0.02 volume sterile cold H2O.  Pellet at 4000
x g at 4°C for 15 minutes.

Final  dilution:   Gently  resuspend  0.002-0.003  volume  sterile cold 10%
glycerol  (filter sterilized/NOT autoclaved)  220 µl aliquots (enough for 5
transformation  reactions  of  40  µl each) should be frozen at -70°C until
needed.   Thaw  on  ice  for  5-10  minutes before use.  When I switched to
filter-sterilized  10%  glycerol, my transformation efficiency for DH5alpha
cells increased to 1.2 e 10 cfu/ug.

Tim Fitzwater
Principal Research Associate
Gilead Sciences


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