Making Competent Cells according to D. Hanahan et al. "DNA Cloning Techniques".
This technique is a little bit more involved than the CaCl2
method but I have found that it yeilds cells that store well and are at
higher competency. If you are looking for a very simple method, use
the CaCl2 method as described in Maniatis or in Current Protocols.
Things to do ahead of time (recipes for some solutions are provided
at the end).
Preparing competent cells.
Prepare 200 ml of sterile SOB media in a 1 liter flask. The instructions
for preparing this are provided on page A.3 of the third volume of the
Maniatis method book or a slightly different recipe is provided at the
end. The sterile 2M MgCl2 solution should be made by diluting
the concentrated lab stock with water. MgCl2 readily absorbs
moisture from the atmosphere so we generally dissolve the entire bottle
of salt at a concentration of 4 M. Also autoclave a test tube for
use in growing a 3 ml starter culture.
Prepare sterile RF1 solution. You will need 67 ml of this.
RF1 consists of 100 mM KCl, 50 mM MnCl2, 30 mM Potassium acetate,
10 mM CaCl2, 15% glycerol. (Note that you are to use Manganese
Chloride and not Magnesium Chloride in RF1.) The solution is adjusted
to pH 5.8 with 10 % glacial acetic acid (do not use the concentrated acid
stock as it is very easy to overshoot the pH adjustment). Filter
sterilize this solution through a pre-rinsed 0.22 micron filter and store
at 4oC. Prerinsing is done by passing water through the
filter (discard this wash). It removes wetting agents often incorporated
into the filter that might inhibit formation of competent cells.
Prepare sterile RF2 solution. You will need 16 ml of this.
RF2 consists of 10 mM MOPS, 10 mM KCl, 75 mM CaCl2, 15% glycerol.
The solution is adjusted to pH 6.8 with 100 mM NaOH and filter sterilized
through a pre-rinsed 0.22 micron filter. Store at 4oC.
Thoroughly clean six 40 ml tubes. Scrub with soapy water and then
rinse with copious amounts of water. Finally, rinse and sterilize
the tubes with ethanol (the 95% stuff in a squirt bottle will do).
It is important that the tubes be free of soap since this reduces the competency
of the cells. It is also important that the tubes be free of contaminating
DNA as this can lead to transformation of the cells. Drain off the
excess ethanol, air dry, cover the tops with foil and set them aside until
Transfer 3 ml of sterile SOB from the 1 liter flask to a sterile test tube
and inoculate with the desired bacteria. Grow up an overnight culture
by shaking at 37oC. Also transfer 1 ml of SOB to a test
tube. This will be used for the blank on the spectrophotometer when
you a tracking the growth of the culture.
The next day, use 1 ml of the overnight to inoculate the SOB that remains
in the 1 liter flask. Incubate at 37oC with shaking.
After 2 hours, start checking the OD550. When the OD550 is between
0.35 and 0.6 for a recA- strain (or between 0.2 and 0.4 for a recA+ strain),
chill the cells on ice. Note, for recA- strains, the OD range corresponds
to 5-9 x 107 viable cells/ml.
Transfer the cells to chilled 40 ml tubes and collect cells by centrifuging
at 3000 rpm for 15 minutes at 4oC in an SS34 rotor.
Pour off the supernatant and remove residual supernatant with a pipette.
Resuspend the cells in a total volume of 67 ml of RF1. Use gentle
vortexing to resuspend the cells. Combine the suspensions of cells
into two tubes and incubate the cells on ice. If the cells are DH5alpha,
incubate for 15 minutes. If the cells are HB101, incubate for 2 hours.
Other cells should probably incubated for 30 minutes although the optimizing
the time would require a systematic analysis.
Collect the cells by centrifuging at 3000 rpm for 15 minutes at 4oC
in an SS34 rotor. Remove as much supernatant as possible and resuspend
the cells in 16 ml of RF2. Incubate the cells on ice for 15 minutes
and then dispense in 400 ul aliquots. Flash freeze in liquid nitrogen
or a dry ice/ethanol bath. Store cells at -75oC.
Add DNA to 100 ul of competent cells.
Incubate on ice for 30 minutes.
Heat shock for 2 minutes at 42oC.
Add 900 ul of LB and incubate for 1 hour at 37oC.
Briefly spin down the cells and remove all but 100 ul of media.
Resuspend the cells and plate on media containing the appropriate antibiotic.
|Component (final concentration)
||Amount for 150 ml
||Component (final concentration)
||Amount for 50 ml
|100 mM KCl (74.55 mw)
||10 mM MOPS pH 6.8 (209.3 mw)
|50 mM MnCl2-4H2O (197.91 mw)
||10 mM KCl (74.55 mw)
|30 mM potassium acetate (98.14 mw)
||75 mM CaCl2-2H2O (147.02 mw)
|10 mM CaCl2-2H2O (147.02 mw)
||15 % glycerol
||Adjust to pH 6.8 with 1 M NaOH and filter sterilize as
|Adjust to pH 5.8 with 10% glacial acetic acid, and filter
sterilize through a pre-rinsed 0.22 micron membrane.
||Amount per 100 mls
||1 ml of 1M stock
||250 ul of 1 M stock
|Adjust pH to 7.0 and autoclave.
||1 ml of 2M sterile stock
||1 ml of 2M sterile stock