DH5α Competent Cells

GroseLab:Protocols

Protocol for Preparation of DH5α Competent Cells

Day 1

Step 1

Streak DH5α for isolation on LB plate (no ampicillin).

Step 2

Grow at 37 ˚C.

Day 2

Step 3

At the end of the day, add a single colony to a 4-16 ml (4 ml for 1X, 16 ml for 4X) SOB media aliquot for overnight culture at 37 ˚C and 250 rpm.

Day 3

Step 4

Add 4.0 mls overnight culture to 400 ml SOB in 2L flask. Grow at 37 ˚C and 250 rpm until OD550 = 0.4-05 (2-3 hours). Transfer to conical 250 ml Tubes.

Step 5

Chill 5-10 min on ice.

Step 6

Spin at 3-4k for 10 min. Pour off sup.

Step 7

Resuspend cell pellet by pipetting in 150 ml TFB I. Ice for 5 min

Step 8

Spin at 3-4k for 10 min at 4 ˚C. Pour off sup.

Step 9

Gently resuspend in 15 ml TFB II (if > 1X pool together, e.g. 60 ml for 4X). Ice for 15 min

Step 10

Aliquot 50 ul to prechilled sterile 0.5 ml microcentrifuge tubes. Snap freeze in matrix of dry ice crushed to powder. Bacteria are ready for transformation.

Media/Buffers

SOB Media

Reagent/Final Vol 400 ml 800 ml 1600 ml Tryptone 8 g 16 g 32 g Yeast 2 g 4 g 8 g NaCl 0.2 g 0.4 g 0.8 g 0.25 M KCl (add after dissolving the foregoing) 4 ml 8 ml 16 ml pH 7.0 with 5 N NaOH. Autoclave. Add 2 mls filter sterilized 2M MgCl2 to each 400 ml.

TFB I

Reagent F.W. 200 ml 400 ml 800 30 mM Potassium acetate 98.14 0.59 g 1.18 g 2.36 g 100 mM RbCl 120.9 2.42 g 4.84 g 9.68 g 10 mM CaCl2 147 0.294 g 0.59 g 1.18 g 50 mM MnCl2 197.9 1.98 g 3.96 g 7.92 g 15% Glycerol N/A 30 mls 60 ml 120 ml pH 5.8 w/ Acetic Acid (do not overshoot—correcting with KOH will cause irreversible precipitation). Filter sterilize.

TFB II

Reagent F.W. 100 ml 10 mM MOPS 209.3 0.209 75 mM CaCl2 147 1.102 10 mM RbCl 120.9 0.121 15% Glycerol N/A 15 ml pH 6.5 w/ KOH. Filter sterilize.

0.25 M KCl (F.W. = 74.55)

1.86 g KCl/100 ml H2O. Filter sterilize.

2M MgCl2 (Hexahydrate = F.W. 203.3)

40.66 g MgCl2 to 100 ml. Filter sterilize.