E. coli Competent Cell Prep
For HB101, DH5, XL1-Blue
(Molecular Cloning Manual 2nd ed. 1989. vol. 1)
1. Inoculate 10 ml O/N culture from a plate or frozen stock. From the O/N culture,
inoculate 0.5 ml into 50 ml LB (in a sterile 250 ml flask). Incubate at 37oC until the
OD600=0.6. This will take 2 - 4 hrs.
2. Cool the cultures by storing on ice for approx. 10 min. Harvest by centrifugation in a
sterile tube at 4000 rpm/10min/4oC.
3. Decant the medium from the cell pellets. Resuspend each pellet in ice-cold 0.1 M
CaCl2 or TFB (10 ml/pellet from 50 ml culture). Incubate on ice for 10 min.
4. Centrifuge cells at 4000 rpm/10 min. at 4oC. Gently resuspend cells in ice-cold CaCl2
or TFB (2 ml/50 ml original culture) and store at 4oC.
Notes
The use of TFB may increase transformation efficiency.
The efficiency of transformation increases during the first 12-24 hours of storage, then
decreases to the original level.
Glycerol (final concentration = 15%) can be added to the cells for storage at -70oC,
although the transformation efficiency will decrease.
Transformation
(Adapted
from a Stratagene protocol accompanying their Epicurian Coli SCS1 (Cat
#200231) competent cells)
1. Aliquot 100 ul competent cells into a pre-chilled 15 ml Falcon 2059 polypropylene
tube (Or equivalent; other tubes may be degraded by the BME. In addition, the heat
pulse step is critical and has been calculated using the heat transfer properties of
falcon tubes. A defined "window" of 45 - 50 seconds is necessary for optimal
transformation efficiencies). If competent cells have been frozen, thaw on ice and
mix gently before transferring to the falcon tubes..
2. Add 3.4 ul of a fresh 1:10 dilution of BME (B-Mercaptoethanol) diluted in high-quality
water, to the 200 ul cells.
3. Swirl gently. Incubate on ice for 10 minutes, swirling gently every 2 minutes.
4. Add 0.1 - 50 ng DNA to the cells and swirl gently. Incubate on ice for 30 minutes.
Heat pulse for 45 seconds (this step is very critical; see step 2) in a 42oC water bath.
Incubate on ice for 2 minutes.
5. Add 800 ul SOC broth (preheated to 42oC) and outgrow the cells for one hour at 37oC,
shaking gently.
6. Plate an appropriate amount of cells on LB agar plates (with the appropriate antibiotic).
Incubate the plates at 37oC O/N.
Notes
To determine the transformation efficiency, transform with 1 ng supercoiled plasmid
DNA and plate 10 and 100 ul.
For subcloning, concentrate the cells after the outgrowth (centrifuge at 4K/2 min in a
microfuge and resuspend in 100 ul SOC). Plate 10 ul and 90 ul.
Buffers and Media
0.1 M CaCl2
(500 ml)
7.35 g CaCl2•2H20
Autoclave
1 M MES (2-[N-morpholine]ethanesulfonic acid)
Dissolve 19.52 g MES in 80 ml ddH2O
Adjust pH to 6.3 with 5 M KOH and bring to final volume of 100 ml
Filter sterilize through 0.45 micron filter, and store at -20oC in 10 ml aliquots
TFB
Reagent
1 M MES (pH 6.3)
MnCl2•4H2O
CaCl2•2H2O
KCl
Hexamminecobalt chloride
Amount/litre
10 ml
8.91 g
1.47 g
7.46 g
0.8 g
Mix all components in 800 ml ddH20
Adjust volume to 1 litre and filter sterilize
Freeze at -20oC or store at 4oC in 40 ml aliquots
The pH of the solution should be between 6.0 and 6.1
SOC Broth (1 litre)
20 g Bacto-Tryptone
5 g Yeast Extract
0.5 g NaCl
Autoclave
Before use add:
(per 100 ml SOB)
1 ml MgCl2/MgSO4
2 ml sterile 20% glucose
MgCl2/MgSO4 (100 ml)
12 g MgSO4
9.5 g MgCl2.
Filter sterilize.
20% Glucose (100 ml)
20 g glucose (dextrose)
Autoclave or filter sterilize
Final concentration
10 mM
45 mM
10 mM
100 mM
3 mM