Making Competent Cells (DH5 cells)
Day 1:
Make 3ml LB culture of DH5 cells with LB and nalidixic acid [not ampicillin]. The colony
should be obtained from streaks made only a few days ago (old cells are not good for making
competent cells). Do not forget the controls.
 No of tubes = 2, amount per tube = 3ml, (2 * 3ml = 6ml)
M1V1 = M2V2
(15mg/ml) V1 = (15l/ml) (10ml)
V1 = (15l/ml) * (10ml) / (15mg/ml)
V1 = 10 l of Nalidixic acid
Put the following at 4C:
 set of 1.7 ml microcentrifuge tubes
 10% glycerol (autoclaved)
 100ml graduated cylinder
 2-4 Sorvall bottles
Put the following at 37C:
 SOB Media
Day 2:
1.
Aliquot 1ml SOB into a cuvette and cover with parafilm. This is the blank. Handle the
cuvette along the side where lines are seen, no fingerprints etc should be present on the
clear side as that would interfere with the reading of the absorbance.
2.
Incubate SOB with 1:500 dilution of the overnight culture.
3.
Turn on the centrifuge and set it at 4C.
4.
Grow culture in an air shaker at 350rpm (300-350rpm) and set temp to 37C (leave the
foil on the flasks).
5.
Incubate till the OD600 reaches 0.6 - 0.8 (approx 4 hours). After 3.5 hours take the OD, to
check the progress of the culture.
6.
Get a bucket of ice. When the OD600 reaches desired level (cells are near the end of log
phase), put the flasks on ice to decrease the temperature to slow cell growth.
7.
Pour culture into chilled Sorvall tubes. Balance them and spin at 7000 rpm for 15
minutes. Cells form a pellet, discard the supernatant. Repeat if there is more culture.
8.
Take 100ml of chilled 10% glycerol. Aliquot 10ml 10% glycerol and resuspend the cells
then add the remaining glycerol.
9.
Centrifuge 7000 rpm for 20 minutes. Discard the supernatant and resuspend the pellet in
100ml of 10% glycerol.
10.
Centrifuge at 7000 rpm for 20 minutes. Discard the supernatant.
11.
Resuspend the cells in what is left . (if it is difficult, then add a max of 2ml of 10%
glycerol).
12.
Aliquot them in 1.7ml microcentrifuge tubes (from 4C) and store them in -80 C (make
sets of approx. 150-250 ml).