Preparation of Competent
E. coli Cells
Add to 100ml of LB media in a 250ml flask, 1ml of overnight E. coli XL1
Blue (from a colony picked from a TET containing plate) grown in the presence
of 12.5µg/ml tetracyclin in LB.
Grow at 37°C, shaking until the A550 is between 0.4 and 0.6. To measure
this, blank the spectrophotometer with a sample of fresh LB and determine
the absorbance of aliquots of culture (maintaining sterility) at half hourly
When the appropriate culture density is reached (this should be within
4 hours), divide into two sterile 50ml universals and chill on ice for
10min and then spin in the Jouan (ie at 4°C) at 2000rpm for 10min.
Meanwhile chill sterile 100mM CaCl2 (made fresh from frozen1M stock and
Resuspend each bacterial pellet in 15ml of ice cold 100mM CaCl2(ie a total
of 30ml for 100ml of culture), with gentle shaking and brief vortexing.
Pool the cells. Keep the temperature below 4°C. Leave on ice for 30min.
Repeat the centrifugation step and resuspend the pellet, now composed of
all of the cultures, in 8.5ml of ice cold 100mM CaCl2.
Leave on ice for a further 30min. The cells are now ready to use. Greater
competency can be obtained by leaving cells on ice for longer periods-
up to 2hours-but this is generally unnecessary for routine subcloning.
To store the cells add 1.5ml of ice cold sterile glycerol, mix gently but
thoroughly, aliquot into 200µl portions in sterile microtubes and
store at -70°C. Test the competency of the cells by transforming a
tube with 1ng of plasmid and look for at least 1000 colonies per ng. Transformation
procedure involves adding a known amount of plasmid (1-10ng) to freshly
thawed competent cells on ice and leaving for 30min. Heat shock for 1.75min
in a 42°C waterbath and add 1 ml of LB media. Incubate at 37°C
with gentle shaking for 1 hour and plate 10µl on an LB/ampicillen/X-gal/IPTG
miniplate (Select plate). Incubate overnight at 37°C and count the
colonies. A colony count of 1 to 10million per µg of input plasmid
is OK, with no white colonies.
Select plates; 100µg/ml ampicillen in cooled LB agar, pour plate
(25ml to 90mm petri dish) and when set spread a mixture of 50µl 20mg/ml
X-gal in DMSO and 50µl 1M IPTG in water (filter sterilised).
pBluescript plasmids are not very good at going blue; incubation at
4°C for an hour followed by a couple of hours back at 37°C sometimes
inproves colour. If things still really arenít turning blue then incubation
at 42°C can help.