One step transformation
kang at msvax.mssm.edu
kang at msvax.mssm.edu
Wed Apr 26 14:55:48 EST 1995
This method was reported by Nishimura et al. 1990 (Nucleic Acid
Res. 18(20) 6169). Theoretically it is same as Chung's method but
modified a little.
I also modified this method a little. Following is mine.
1. To the 100 ml of LB add 1 ml of 100X MG solution and 1 ml of
overnight culture of bacteria; 100X MG: 1M MgSO4, 20% glucose
(W/V). The bigger flask is the better.
2. Incubate 4 hours at 37oC with maximum speed shaking.
3. Pour culture to the prechilled two Falcon 50 ml tubes.
4. Leave the tubes in the ice bucket for 30 min.
5. Centrifuge with minimum G force (1500 rpm with Soval ss-34).
6. Pour off supernatant, but not completely.
7. Resuspend the pellet with gentle tapping. You don't have to add
extra solution cause there remains some solution in the tube.
8. Add 2.5 ml of ice cold storage solution; Storage soln: 36% glycerin
(W/V), 12% PEG (W/V; I am using 7,000 but 6,000 or 8,000 maybe O.K.),
12 mM MgSO4 in LB.
9. Aliquot 0.1 ml to each tube.
10. Keep in -70oC deep freezer.
If you follow this protocol you will easily have at least 5X10^6
competent cell. However, If you want to get higher efficiency
competent cell you should be a little more careful. Following is the
point I want to stress.
1. The culture should be aerobic. This is the reason why I described
the bigger flask and maximum speed shaking. If possible I
recommend to use fermentor with air supply.
2. The incubation time after inoculation may vary depend on strain
to strain. The 4 hour used by myself is for XL-1 cell line.
3. The LB should be warmed 37oC before inoculation.
4. 0.9 ml of SOC not LB should be added after heat shock of
competent cell; I do not know the exact reason but SOC increases the
competency 5 to 10 times higher than LB.
5. I tested TB for culture medium but the competency was 1/5 of LB.
(I thought increased growth ratio will increase competency but it
6. Nishimura mentioned higher growth rate because of glucose will
increase the competency but I found that increased glucose
concentration (up to 10 %) does not give any difference to me.
7. Using detergent free system is mandatory (Autoclave your
glassware filled with ddH2O and throw out before culture). I
designated 1 Erlenmeyer flask only for this purpose and never
washed with detergent.
8. Instead of aliquoting 0.1 ml you can aliquot 1 ml and you can
aliquot it 0.1 ml after -70oC freezing and thawing. It will save some
of your freezer space but not much time or labor. The competency seems to
remain same as original even after 2nd freezing and thawing.
9. I did not add DMSO for this system. If someone find that is good
please e-mail me.
10. After I set up this method I never went back to CaCl2, Dr.
Hanahan's method and even to electrophoration. Electrophoration
seems to give same or a little higher competency than this methods
but it is cumbersome to repeat centrifuge and wash several times.
Not to saying about the price of instrument and cuvet.
11. Frankly speaking, if you can not get more than several hundred
colonies from just simple subcloning ( I usually use 40 ng insert and
vector for 10 ul ligation mix and use 1 ul of rexn mix for
transforming 100 ul of this competent cell. If I plate 200 ul out of 1
ml cell and SOC mix usually I get 3X10^3 colonies), the problem does
not lie on your competent cell but lie on your DNA or ligase. I know
very pity peoples who are purchasing 10^9 competent cell just for
subcloning. It is really ridiculous.
I want to heare another fancy modification from you.
Wizard of TM (transgenic mice).
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