Streptococcus transformation protocol

Culture media:

1.     For overnight culture (SGM17):

Beef extract ………………..    5.00 g/l

Polypeptone ………………     5.00 g/l

Phytone-peptone ……………  5.00g/l

Yeast extract ………………..  2.50 g/l

Disodium glycerol phosphate 19.00 g/l

Ascorbic acid ………………...0.50 g/l

Glucose ………………………5.00 g/l

MgSO4.7H2O ………………. 0.25 g/l

+

0.5 M Sucrose (171.15 g/l)

-    Filter-sterilize                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

2.     For growing the cells to make electrocompetent cells:

SGM17 (above) + 0.5% glycine

3.     For recovery after electroporation:

SGM17 containing 20 mM MgCl2 and 2 mM CaCl2

4.     For plating:

SR medium contains (g/l): (Supplement with erythromycin (5 and 10 mg/ml)

-       Tryptone         10

-       Yeast extract   5.0

-       Sucrose           200

-       glucose            10

-       Gelatin,           25

-       agar                 15

-       MgCl               2.5 mM

-       CaCl₂              2.5 mM

*Pour plates in the anaerobic chamber.

Method:

Day1. Grow Streptococcus in 10 ml of GM17+Sucrose culture medium overnight aerobically.

Day2. Preparation of competent cells

-       Inoculate prewarmed 250 ml of GM17+Sucrose +0.5% glycine culture medium with 5 ml of the overnight culture.

-       Grow aerobically at 37 °C to an OD600 of 0.2 (approximately 5 to 7 h).

-       Place the culture on ice for 30 minutes and centrifuge at 5000 g, 4 °C, for 15 min.

-       Wash the cell pellets three times in 25 ml of ice-cold 0.5 M sucrose/10% glycerol, and centrifuge as before.

-       Resuspend the cell pellet in 0.5 ml of 0.5 M sucrose/10% glycerol and either use immediately for electroporation or store at -80 °C.

Electroporation:

-       Mix 100 ml of the cell suspension was mixed with up to 5 ml of DNA and place in ice-cold 0.2-cm electroporation cuvette (Bio-Rad Laboratories, Richmond, CA).

-       Electroporate at 2.5 kV, 200 W, 25 mF using the Bio-Rad Gene Pulser.

-       Immediately after electroporation, add 0.9 ml of SGM17 containing 20 mM MgCl2 and 2 mM CaCl2 to the cell suspension.

-       Transfer to a tube and incubate the mixture at 37 °C for 1 h.

-       Dilutions of the cells were plated on SR and SDM agar plates supplemented with antibiotic (5 mg/ml erythromycin).

-       Incubate in the anaerobic chamber and incubate at 37 °C for 1-2 days.