Team:Fatih Turkey/Procedures

MATERIALS

·         Centrifuge

·         Autoclave

·         Incubator with shaker

·         pH meter

·         Stock competent cell

·         LB broth

·         Falcon

·         Ice

·         Liquid nitrogen

·         Bacto yeast extract

·         Bacto tryptone

·         Magnesium sulfate

·         Potassium hydroxide

·         Potassium acetate

·         Rubidium chloride

·         Calcium chloride

·         Manganese chloride

·         Glycerol

·         Dilute acetic acid

·         Filter

·         MOPS

·         Dilute NAOH

SOLUTIONS

Preparation of Psi broth (per liter)
- 5 g bacto yeast extract
- 20 g bacto tryptone
- 5 g magnesium sulfate
- PH 7.6 with potassium hydroxide 
- Autoclave 40 min

Preparation of TfbI (per 200 ml)
- 0.588 g potassium acetate (final molarity/conc= 30 mM)
- 2.42 g rubidium chloride (final molarity/conc= 100 mM)
- 0.294 g calcium chloride (final molarity/conc= 10 mM)
- 2.0 g manganese chloride (final molarity/conc= 50 mM)
- 30 mL glycerol (15% v/v)
- Adjust PH 5.8 with dilute acetic acid
- Sterilize with filter 

Preparation of TfbII (per 100 ml)
- 0.21 g MOPS (final molarity/conc= 10 mM)
- 1.1 g calcium chloride (final molarity/conc= 75 mM)
- 0.121 g rubidium chloride (final molarity/conc= 10 mM)
- 15 mL glycerol (15% v/v)
- Adjust PH 6.5 with dilute NaOH 
- Sterilize with filter

- Inoculate streak plates from liquid stock competent cells and incubate overnight at 37˚C

- Put 10 mL LB + colony  into 50ml falcon. Incubate overnight at 37˚C with shaker

- Inoculate 200 ul to 1000 ul from overnight culture into 100-500 ml Psi broth  (scale up or down as needed). Incubate at 37 C with aeration to A600=0.6-0.7

- Ice 15 min.  From this step onward the cells must remain COLD. (4C or on ice)
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
- Discard supernatant and add 0.4 volume (ie of original volume, here it is 40-400 ml) TfbI, resuspend and ice 15 min.
- Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
- Discard supernatant and resuspend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 100ul  to 200ul aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.