TFP1

100 mM RbCl

50 mM MnCl2

30mM potassium acetate

10mM CaCl₂

15%glycerol

TFP2

100 mM MOPS

50 mM RbCl

75 mM CaCl₂

15%glycerol

SOC medium

Tryptophane 20g/L

Yeast extract 5g/L

NaCl 0.5g/L

250mM KCl

1M MgCl₂

50% (w/v) sterile glucose

Filter the solution through a 0.2 µm filter.

3T3 medium

D-MEM 1x: 500 mL

FBS 100%: 50 mL

GlutaMax 100x: 5 mL

Penicillin (10k units/mL) /Streptomyocin (10k µg/mL ): 5 mL

Jurkat medium

MEM 1x: 2 mL

FBS 100%: 20 mL

GlutaMax 100x: 2 mL

Penicillin (10k units/mL) /Streptomyocin (10k µg/mL ): 0.4 mL

RPMI: 200 mL

Cryostock of bacteria

• Add 1 mL of 40% glycerol to a cryogenic vial.
• Add 1 mL sample from the bacterial culture.
• Vortex the vial.
• Store in -80ºC freezer.

Preparation of chemically competent cells

• Take a trace of Escherichia coli from a glycerol stock vial and put it on a LB plate with the correspondent antibiotic.
• Incubate overnight at 37ºC.
• Pick a colony and inoculate 10mL LB medium containing the relevant antibiotic. Grow overnight at 37ºC.
• Add 1mL overnight culture to 100mL prewarmed LB medium containing the relevant antibiotic in a 500mL flask, and shake at 37ºC until an OD₆₀₀ of 0.5 is reached (usually 90-120 min).
• Cool the culture on ice 5min, and transfer the culture to a sterile, round-bottom centrifuge tube.
• Collect the cells by centrifugation at low speed (5 min, 4000 xg, 4ºC).
• Resuspend the cells gently in cold (4ºC) TFB1 buffer (30 mL for 100 mL culture) and keep the suspension on ice for 90min.
• Collect the cells by centrifugation (5min, 4000 xg, 4ºC).
• Discard the supernatant carefully. Always keep the cells on ice.
• Resuspend the cells carefully in 4 mL ice-cold TFB2 buffer
• Prepare aliquots of 100-200µL in sterile microcentrifuge tubes and freeze in dry-ice-ethanol mix. Store the competent cells at -70ºC

Transformation of chemically competent cells

• Take 1 µL DNA to be transformed and put into a cold sterile 1.5 mL microcentrifuge tube, and keep on ice.
• Thaw an aliquot of frozen competent cells on ice.
• Resuspend the cells and transfer 50 of the cell suspension into the microcentrifuge tube with the plasmid DNA, mix carefullu, and keep on ice for 20 min.
• Transfer the tube to a 42ºC water bath for 90 s.
• Add 500 µL SOC medium to the cells and incubate for 60-90 min at 37ºC. Shaking increases transformation efficiency.
• Plate 100 µL on LB-agar plates containing antibiotics. Incubate the plates overnight at 37ºC.