TRANSFORMATION OF BACTERIA |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. Luria-Bertani (LB) media (1 L): | Mix 10 g
of Bacto-tryptone, 5 g of Yeast extract, and 10 g of NaCl,. pH to 7.5 w/ NaOH. Add dH2O to 1 L (Autoclave) |
| 2. LB plates: | Mix 500 mL of LB media with 7 g of Agar. Autoclave. Cool to ~55-65oC prior to pouring. The addition of antibiotics should be made before pouring
and at a temperature not higher than 55 oC. Antibiotics can also be spread on previously made plates,
but this is not very effective (unequal absorption, etc...)
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| PROCEDURE | |
| 1. First prepare competent cells (Preparation of competent E. coli cells using CaCl2) (or purchase them) | |
| NOTE: instead of LB, SOC media is a popular nutrient media for these applications: the recipee is as follows: Tryptone 16g (Difco #0123-05-3), Yeast Extract 4g (Difco #0127-05-3), NaCl 0.4g (Fisher #S671-500;). Make up to 800 ml in dH2O. Swirl to dissolve, then autoclave. Before use add MgCl2 (10 mL/L of 1M MgCl2) and MgSO4 (20 mL/L of 1M MgSO4; note: require 20 mM MgSO4 for this SOB). | |
| 2. Remove competent cells from -80oC. Thaw competent cells (140 ml in a tube) on ice, and pippet ~2-10 mL of ligation reaction, directly to the cold tube (diluting the ligation reaction >5 fold prior to adding to competent cells may help in the transformation -it aids in the equal spread of the DNA) | |
3. Mix all reactions by tapping the tubes, and incubate on ice for 30 min |
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| 4. Heat shock cells (for 60-120 sec in a 42oC water bath), cool on ice (5 mins) | |
| 5. Add 800 mL of LB, to each reaction and mix (other media can also be used: SOC, YT, TB, etc... LB for the most part will give the same results and it will not make a big difference in making the transformation of the ligation reaction any more efficient) | |
| 6. Incubate on shaker (250 rpm) at 37oC for 1 hour to allow the expression of the antibiotic resistance markers (other markers require different lenghts of time: Ampicillin requires 1 hour or less, Kanamycin requires an incubation of 2 hours, ...) | |
| 7. Pipete 50-200 mL of each transformation on LB+ antibiotic plates and spread cells with bent glass rod | |
8. Place plates inverted at 37oC for at least 18 hours |
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| 9. Place plates at 4oC until picking colonies (colonies should remain viable for about 1-2 months) | |
| NOTE: If using blue/white selection (b-galactosidase), place plates at 4oC to help in color development | |