TRANSFORMATION OF BACTERIA
 
Last Update: December 2006
 
 
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...)
   
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

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

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