TG1 Competent Cells

1. Streak out E. coli TG1 cells with an inoculation loop onto a Minimal Medium plate.

2. Incubate the plate overnight at 37°C

3. Pick a single colony of E. coli TG1, and inoculate 15 ml of 2X TY in a 100 ml flask.

4. Incubate the flask overnight on a shaking platform (275 rpm) at 37°C.

5. Prepare two 2 liter baffled flasks containing 500 ml of 2X TY Media each. Inoculate each 2 liter flask with 5 ml of the overnight culture from Step #4.

6. Incubate the flasks overnight on a shaking platform (275 rpm) at 37°C. After 1.5 hr, transfer 1 ml of each culture to plastic cuvettes and measure the optical density at 600 nm (OD600). Remove the cultures when the OD600 is 0.5.

7. Transfer the bacterial cultures to sterile, pre-chilled (4°C) 500 ml centrifuge bottles. Balance the bottles and place them on ice for 20 min (see Hint #2).

8. Centrifuge the bottles at 3,000 X g for 15 min at 4°C in a Sorvall™ GSA rotor.

9. Discard the supernatants and resuspend the cell pellets by gentle trituration in 1 liter of ice-cold Hepes Solution.

10. Repeat Step #8.

11. Discard the supernatant and resuspend the cell pellets by gentle trituration in 500 ml of ice-cold Hepes Solution. Pool the cell suspensions in two of the centrifuge bottles.

12. Repeat Step #8.

13. Discard the supernatant and resuspend all the cells by gentle trituration in a total volume of 20 ml of Hepes/Glycerol Solution. Pool the cell suspension into a sterile Sorvall™ SS-34 centrifuge tube.

14. Centrifuge the tube at 3,000 X g for 15 min at 4°C in a Sorvall™ SS-34 rotor.

15. Prepare an Ethanol/dry ice bath .

13. Discard the supernatant from the tube in Step #14 and gently resuspend the cell pellet in a total volume of 2 ml of sterile 10% (v/v) Glycerol.

14. Use the cells immediately for transformation, or freeze 220 μl aliquots of cells in sterile microcentrifuge tubes that have been pre-chilled in the Ethanol/dry ice bath (see Hint #3).

Solutions

1 mM HEPES, pH 7.4

Filter to sterilize

10% (v/v) Glycerol

Prepare just before use Filter to sterilize

Hepes/Glycerol Solution

Prepare just before use

1 mM HEPES, pH 7.4 10% (w/v) Glycerol Filter to sterilize

2 X TY Media

5 g NaCl Prepare in 1 liter of ddH2O

10 g Bacto Yeast Extract (Difco)

16 g Bacto Tryptone (Difco)

Autoclave to sterilize

20% (w/v) Dextrose Filter to sterilize 20% (w/v) D-Glucose

1 % (w/v) Vitamin B1

Filter to sterilize and store at 4°C 1% (w/v) Thiamine-HCl

1 M Magnesium Sulfate 1 M Magnesium Sulfate (MgSO4)

Minimal Media

1 g Ammonium Sulfate ((NH4)2SO4)

10 ml of 20% (w/v)

Dextrose 0.5 g

Sodium Citrate, Dihydrate 15 g

Bacto Agar (Difco)

Add 1 ml of 1 M Magnesium Sulfate

0.5 ml of 1% (w/v) Vitamin B1

4.5 g Potassium Phosphate, Monobasic (KH2PO4)

Pour into petri dishes

Prepare in 1 liter of ddH2O

Autoclave the solution and cool to 50°C

10.5 g Potassium Phosphate, Dibasic (K2HPO4)

Transformation

add 24 mM of beta-mercaptoethanol to the transformation mixture, 10 minutes before DNA addition, the transformation efficiency can be increased by 10 folds

Protocol 2

Centrifuge at 1200g, 15 min, 4°C.
10. Resuspend the pellets with a 10-mL pipet in cold 10% glycerol to a total
volume of 250 mL and transfer to one centrifuge bottle. Keep cells on ice while
resuspending.
11. Centrifuge at 1200g, 15 min, 4°C.
12. Remove supernatant with a pipet. Be careful because the pellet is soft here.
Resuspend the pellet with a 10-mL pipet in 10% glycerol to a total volume of
50 mL. Keep cells on ice while resuspending.
13. Centrifuge at 1200g, 15 min, 4°C.
14. Remove supernatant with a pipet. Be careful because the pellet is very soft here.
Resuspend the pellet with a 10-mL pipet in residual glycerol. Keep cells on ice
while resuspending. The final volume should be 5–10 mL/L original culture.
15. Aliquot 200 μL/cryotube and quick-freeze in liquid nitrogen (wear safety glasses
when doing this). Store cells at –80°C.
16. Transformation efficiency of cells should be determined by electroporating 100 pg
control (uncut) vector DNA and plating onto appropriate selective medium (see
Subheading 3.13.). Cells should yield 109 transformants/μg DNA.