- Inoculate a single colony from a rich plate (Luria, Luria-Bertani)
into 2 ml of rich broth (Luria-Bertani; RB) in a plating tube. Shake overnight at 37°C.
- Subculture the overnight 1:100 in 1 Volume Unit of RB+20
mM MgSO4 (typically 250 ml). Grow to OD590=0.4-0.6 or Klett=60 (~2-3 h).
- Centrifuge 5,000 rpm 5 min at 4 C. For 250 ml culture,
use 2 250 ml centrifuge bottles, large rotor.
- Gently resuspend pellent in 1/2.5 Volume Unit ice cold
TFBI. For 250 ml subculture, use 100 ml TFBI; 50 ml/bottle. Combine the resuspended cells in one
bottle. Keep all steps on ice and chill all pipets, tubes, flasks, etc. from this point on.
- Incubate on ice for 5 min.
- Centrifuge 5,000 rpm 5 min 4°C
- resuspend pellet in 1/25 original volume cold TFB2. For
250 ml of original subculture, use 10 ml TFB2.
- Incubate on ice 15-60 min. before aliquoting 100 ul/tube
for storage at -70°C. Quick-freeze the tubes. A convenient way to do this is to use ice-bath
racks. These have a movable "lid" rack, with an ice compartment bottom (American Scientific
Products, cat # S9233-1); set up the top-labeled tubes (open) in a rack with ice in the bottom compartment;
distribute the cells; then close the tubes. In another bottom compartment, set up a dry ice/ethanol
(or isopropanol) bath, wait for it to stop bubbling, then transfer the "lid" rack (which
carries the tubes) to the dry ice bath bottom compartment for ~15 sec; drain the isopropanol, wipe
with a tissue to get rid of the isopropanol, transfer to an empty bottom compartment and put the
whole thing in the -70 freezer. After the tubes are well-frozen they can be dumped loose into a box
or ice-cream carton, or transferred to slots in a storage box. Be careful not to get alcohol on the
lips of the tubes. Liquid nitrogen can also be used, but not with these racks.
- To transform, thaw an aliquot on ice; add DNA; incubate
1 h on ice; heat shock 45 seconds at 37°C; incubate on ice 2 min; dilute 15-fold into RB with
no drug (for phenotypic expression); grow with vigorous aeration at 37°C for 20 min.; plate on
selective medium.
This procedure works with most strains and should routinely
give > 107 cfu/ug
of pBR322 with reasonably healthy K-12 derivatives (using 0.1 ng/transformation). Frozen cells last
at least a year.
Recipes
RB (Luria-Bertani medium)
per liter:
10 g Tryptone (Difco)
5 g Yeast Extract (Difco)
5 g NaCl
2 ml 1N NaOH
TFBI
30 mM KOAc (potassium acetate)
100 mM RbCl
10 mM CaCl2
50 mM MnCl2
15% glycerol
Adjust to pH 5.8 with acetic acid and filter (0.45 um, Nalgene
units or Millipore filters) to sterilize.
It is convenient to make this as:
| 5 g |
RbCl (Alfa) |
| 12.3 ml |
KOAc 1 M |
| 4.1 ml |
CaCl2 1 M |
| 20.5 ml |
MnCl2 1 M (this is pink) |
| 61.5 g |
glycerol |
| pH to 5.8 with |
8 ml HOAc 0.1 M |
make up to 410 ml; distribute in 100 ml sterile aliquots;
and use 1 aliquot/250 ml culture.
TFBII
10 mM MOPS or PIPES
75 mM CaCl2
10 mM RbCl
15% glycerol
Adjust pH to 6.5 with KOH and filter to sterilize
Make up as
| 1.5 ml |
MOPS 1 M pH 6.5 (this is yellow) |
| 11.25 ml |
CaCl2 1 M |
| 1.5 ml |
RbCl 1 M |
| 22.5 g |
glycerol |
| pH with |
1 N K0H |
make to 150 ml; filter; use 10 ml per original 250 ml culture.
Procedure adapted from the John Innes Institute
(Norwich, England) via Joseph Utermohlen (Univ. of Arizona).
This procedure improves transformation
with most strain backgrounds, when compared with CaCl2 procedure.
Note that Dam- strains (e.g GM2163)
will yield 10-100-fold fewer transformants than isogenic Dam+ strains, probably due to poor initiation
of the second round of replication. BL21(DE3) also transforms poorly with this and other transformation
procedures.
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