(Inoue, Gene 96 (90) 23-28)
1) Pick a few colonies from plates and inoculate into 100 ml of SOB medium in 500 ml flask. Grow at 18 °C with vigorous shaking (the cells may be grown at 37 °C for 1-2 hours initially before lowering the temp. to 18 °C).
2) When the OD600 reached 0.6 (takes < 30 hours), place on ice for 10 min., then spin down cells at 2000 g for 10 minutes.
3) Remove supernatant and resuspend gently in 30 ml of ice-cold TB. Incubate on ice for 10 min.
4) Spin down cells at 800 - 1000 g and resuspend pellet gently in 8 ml TB (you may notice a change in the cells that makes them easier to spin down and resuspend). Add DMSO to a final concentration of 7%. Incubate on ice for further 10 min.
5) Aliquot cells and freeze immediately in liquid N2.
* TB - 10mM Pipes, 15mM CaCl2, 250mM KCl, pH to 6.7 with KOH, then add MnCl2 (55mM final concentration). Filter sterilised
Transformation of competent cells
1) Defrost frozen competent cells slowly on ice (~30 min.). Add 200 µl of competent cells to 1-10 µl of DNA solution in tubes, swirling gently. Flick tube a few times and incubate on ice for 30 min.
2) Heat-shock at 42 °C for 30 sec. - 2 min. (time varies with different E. coli strains).
3) Return tubes to ice, then add 0.5-0.8 ml of pre-warmed SOC. Incubate at 37 °C for 30-60 minutes.
4) Plate onto selective LB agar and incubate overnight at 37°C.
Note:
Competent cells are fragile (cell wall is thought to be weakened), therefore treat the cells gently when preparing these cells. Do not vortex or pippette up and down to resuspend the cells. Do not spin the cells at too great a speed (spinning down at 5000g will cause some cells to lyse).
Always keep the cells chilled when making competent cell. Do not let them warm up.
Freezing the cells appear to make cells more competent.
Some cell strains may work better than others (DH5alpha works well in my hand). Note also that some cells (e.g. HB101) has greater recombination activity than others.
This method doesn't appear to work with BL21, so just grow the cells at 30 or 37 °C when making BL21 competent cells. However, it has been suggested that the efficiency of BL21 prepared using Inoue method may be improved by treating it with DTT before freezing (add to 3.5% v/v of a 2.2M DTT, 10mM KAc pH6 solution and incubate 10 minutes on ice).
Heat shock time should be determined for different strains of cell. For DH5alpha or JM109 use 30-45 sec. For BL21 use 120 sec.
Deactivate ligase prior to transformation. Ligase may reduce transformation efficiency.
Diluting the ligation mixture (~5x) can also increase transformation efficiecy by reducing the amount of reagents/contaminants that may affect transformation. Likewise it has been suggested that phenol/chloroform treatment may also increase efficiency, but it is probably too much trouble to bother trying.
The DNA added should not be more then 5% of the volume of competent cells used. The final DNA concentration should not exceed 5 ng/ul.
The method above should give a transformation efficiency of more than 108 cfu per ug of plasmid DNA (pUC or pBluescripts) with over 109 cfu possible.
Transformation efficiency has a roughly inverse relationship with the size of plasmids. Cells with deoR mutaion (e.g. DH5alpha) can improved the transformation of large plasmid. Relaxed plasmids has ~3/4 of the transformation efficiency of supercoiled plasmid.
2 different plasmids can be transformed at the same time, or one after another. But they must be compatible (they cannot have the same replicon).
For routine transformation whereby efficiency of transformation is of no import, some of the steps may be shorten or omitted. For example, heat-shock step may be unnecessary and recovery incubation time at 37 °C can be reduced or omitted (but do note that this may depends on the antibiotic used for selection - for ampicillin-type antibiotics the incubation time is not really that important, therefore you can plate the cells straight after heat-shock if you wish. for other antibiotics, however, the incubation time may be essential).
Plating cells - dry 1.5% agar plates (exposed upside down) at 37 °C for 2-4 hours just before use, the plate should be able to soak up to 0.8-1 ml of media when plating. For blue-white selection, it is not necessary to make X-gal plate, just add X-gal + IPTG direct to cells, mix and then plate.
Detergents may be detrimental to the transformability of the competent cells, therefore the glassware used for making competent cells should not be washed with detergents. Polycarbonate flask may also be used instead of glass flask. DMSO can dissolve polystyrene, therefore use polypropylene tubes.
When cloning difficult and less stable sequence (e.g palindrome, repeats, LTR sequences), it helps to grow transform cells at lower temperatures (25-30 °C or room temperature) in very rich media (e.g. Terrific Broth). Also terminate growth before reaching late stationary growth phase when grown in liquid media (i.e harvest cells at OD550 between 1 and 2). Use of stabilizing strain is also useful.
There are other methods of making competent cells - e.g. CaCl2 method, RbCl method which is more effective than CaCl2 method. Electroporation is supposed to give higher efficiency (up to 1010 transformants per ug plasmid claimed), but for the simple cloning that we do, its use is not warranted (and it's more expensive, more trouble than it's worth, etc.).
If a cooling shaker is not available - grow the cells at room temperature. More discussions on making competent cell as well as references can be found in TIBS articles "Preparing ultra-competent E.coli" and "Better competent cells"
It is also possible to transform cells straight from plate. It is convenient but you should expect low efficiency. See the following reference for more details (as well as more information on competent cells and other protocols):
Hanahan et al, Methods in Enzymology 204, 63
Luria-Bertani (LB) medium
Per litre:
10 g tryptone
5 g yeast extract
5 g NaCl (10g for Miller's LB)
SOB
Per litre:
20 g tryptone
5 g yeast extract
0.5 g NaCl
after autoclaving the above, add
10 ml 0.25M KCl
5 ml 2M MgSO4
100 ul 5M NaOH
SOC
- Same as SOB but with added 20 ml of 1M glucose
TB (Terrific Broth) Medium
For a litre:
Solution I - 12 g tryptone
24 g yeast extract
4 ml glycerol
900 ml distilled water
Solution II - 2.3 g KH2PO4
12.5 g K2HPO4
100 ml distilled water
Autoclave both solutions, cool and mix together.
