Tuesday, August 21, 2007
Preparation of Electroporation Competent Bacteria
The night before:
1. Grow an overnight of the appropriate bacteria- 50 mls per liter of cells. (e.g. TG-1, TG-2, XL-1 Blue, R1L).
2. Prepare 10 % Glycerol solution and cool in the cold room (sterile and autoclaving). Put 2 bottles of sterile dH2O in the cold room.
The morning of preparation:
1. in the morning inoculate 1 L of LB with 50 mls of the overnight. Grow the cells to an O.D.550=0.5. Should take about 2 hours.
2. Place flask in ice-water for 10 minutes to quickly cool cells. Meanwhile, chill two 250 ml conical bottles and 35 sterile Eppendorf tubes on ice (Eppendorf tubes can be chilled in the cold room). Keep everything on ice as much as possible for the remainder of the protocol.
3. Pour the grown culture into the 250 ml bottles, 500 mls of your culture.
4. Spin down the cells at 3000 rpm for 8 minutes at 4oC in RC3B. Pour off supernatant. Add the remaining 500 mls of culture to the bottles and spin again.
5. Resuspend each cell pellet in 10 ml ice cold sterile dH2O using a fresh plastic sterile pipette.
6. Bring each tube to 200 ml with ice cold sterile dH2O. Mix and spin down as above.
7. Repeat steps 5 and 6.
8. Wash each of pellets with 40 ml of ice cold 10% glycerol.
9. Spin down cells and remove supernatant as above.
10. Resuspend each of the pellets in 1.2 mls ice cold 10% glycerol.
11. Aliquot cells (80 ul per aliquot; enough for 2 transformations) into sterile ice cold Eppendorf tubes.
12. Snap freeze aliquots ina dry-ice ethanol bath and place in a pre-chilled box at -70oC.
1. Grow an overnight of the appropriate bacteria- 50 mls per liter of cells. (e.g. TG-1, TG-2, XL-1 Blue, R1L).
2. Prepare 10 % Glycerol solution and cool in the cold room (sterile and autoclaving). Put 2 bottles of sterile dH2O in the cold room.
The morning of preparation:
1. in the morning inoculate 1 L of LB with 50 mls of the overnight. Grow the cells to an O.D.550=0.5. Should take about 2 hours.
2. Place flask in ice-water for 10 minutes to quickly cool cells. Meanwhile, chill two 250 ml conical bottles and 35 sterile Eppendorf tubes on ice (Eppendorf tubes can be chilled in the cold room). Keep everything on ice as much as possible for the remainder of the protocol.
3. Pour the grown culture into the 250 ml bottles, 500 mls of your culture.
4. Spin down the cells at 3000 rpm for 8 minutes at 4oC in RC3B. Pour off supernatant. Add the remaining 500 mls of culture to the bottles and spin again.
5. Resuspend each cell pellet in 10 ml ice cold sterile dH2O using a fresh plastic sterile pipette.
6. Bring each tube to 200 ml with ice cold sterile dH2O. Mix and spin down as above.
7. Repeat steps 5 and 6.
8. Wash each of pellets with 40 ml of ice cold 10% glycerol.
9. Spin down cells and remove supernatant as above.
10. Resuspend each of the pellets in 1.2 mls ice cold 10% glycerol.
11. Aliquot cells (80 ul per aliquot; enough for 2 transformations) into sterile ice cold Eppendorf tubes.
12. Snap freeze aliquots ina dry-ice ethanol bath and place in a pre-chilled box at -70oC.
Flow cytometry_GFP and PI or DNA simotaneously
A good method to fix cell to detect GFP signal:
Fixation reagent:
2% Paraformaldehyde, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 1mM EDTA, 10 mM PIPES, pH 6.8
Dyhydration : 70 % ice-cold ethanol
Procedure:
wash cells with PBS, then Fixation reagent for 30 min, room temperature, PBS wash 4X, then 70% ethanol for 10-12 hours. PBS wash, RNaseA (50ug/ml) for 30 min, PBS wash, PI (50ug/ml) 30 min before FACS analysis.
Reference:
Nucleic Acids Research, V. 25, p 4855-4857.
A rapid, quantitative and inexpensive method for detecting apoptosis by flow cytometry in transiently transfected cells.
Lamm GM, Christofori G.
Fixation reagent:
2% Paraformaldehyde, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 1mM EDTA, 10 mM PIPES, pH 6.8
Dyhydration : 70 % ice-cold ethanol
Procedure:
wash cells with PBS, then Fixation reagent for 30 min, room temperature, PBS wash 4X, then 70% ethanol for 10-12 hours. PBS wash, RNaseA (50ug/ml) for 30 min, PBS wash, PI (50ug/ml) 30 min before FACS analysis.
Reference:Nucleic Acids Research, V. 25, p 4855-4857.
A rapid, quantitative and inexpensive method for detecting apoptosis by flow cytometry in transiently transfected cells.
Lamm GM, Christofori G.
