E. coli Heat Shock

PROTOCOL DUMP PLEASE FORMAT

Preparing Chemical Competent Cells

StreakE. coliDH10B on an LB + Strep50 plate. Grow O/N at 37C.
Pick colony and inoculate in 5 mL LB + Strep50. Grow for 16 +/- 4 hrs in 37C shaker.
After 16 hours, inoculate 300 uL of the overnight culture into 100 mL LB + Strep25 in a flask.
Grow in 37C shaker for about 3 hrs, or until OD = 0.2-0.4.
While cells are growing:* Label 25 microcentrifuge tubes and store at -80C* Make 2x36 mL 0.125 M CaCl2 by combining 4.5 mL 1 M CaCl2 + 31.5 mL autoclaved ddH2O each in 2 50 mL Falcon tubes
Transfer culture to 2 50 mL Falcon tubes and put on ice for 30 min.
After leaving on ice, spin down in tabletop centrifuge at max RPM, 4C, for 10 min.
Decant supernatant. Add 20 mL cold 0.125 M CaCl2.
Gently resuspend cells by repipetting (or shaking -G), and combine the contents of the 2 tubes. Re-centrifuge as above.
Repeat CaCl2 wash as above.
Decant supernatant. Add 0.6 mL 0.125 M CaCl2 and 0.4 mL 50% glycerol and gently resuspend by repipetting. Add cells to pre-cooled microcentrifuge tubes in 55 uL aliquots. Store at -80C.

Transformation

Pre-heat heat block to 42C. Fill wells that you will use with DI water. Fill an ice bucket with ice.
Put CC cells on ice and allow to thaw for ~2 min. Add 1 uL 40% PEG (in 4C) to each 55 uL aliquot of CC cells.
Add 0.5-3 uL plasmid (0.5 uL if they are miniprepped - supercoiled DNA increases transformation efficiency. If plasmid is a ligation product, use 3 uL - this plasmid is not supercoiled, which reduces the efficiency. If the plasmid is a ligation product of a digested plasmid and annealed oligos, the plasmid is nicked, which further reduces transformation efficiency).
Put on ice 30 min.
Put on heat block (in filled wells)1 min, 10 s.
Put back on ice for 2 min.
Add 400 uL SOC.
Put in 37C incubator for 1 hr.
Continue protocol @Plating(see below).

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Last updated 6 years ago

Preparing Chemical Competent Cells

StreakE. coliDH10B on an LB + Strep50 plate. Grow O/N at 37C.

Pick colony and inoculate in 5 mL LB + Strep50. Grow for 16 +/- 4 hrs in 37C shaker.

After 16 hours, inoculate 300 uL of the overnight culture into 100 mL LB + Strep25 in a flask.

Grow in 37C shaker for about 3 hrs, or until OD = 0.2-0.4.

While cells are growing:* Label 25 microcentrifuge tubes and store at -80C* Make 2x36 mL 0.125 M CaCl2 by combining 4.5 mL 1 M CaCl2 + 31.5 mL autoclaved ddH2O each in 2 50 mL Falcon tubes

Transfer culture to 2 50 mL Falcon tubes and put on ice for 30 min.

After leaving on ice, spin down in tabletop centrifuge at max RPM, 4C, for 10 min.

Decant supernatant. Add 20 mL cold 0.125 M CaCl2.

Gently resuspend cells by repipetting (or shaking -G), and combine the contents of the 2 tubes. Re-centrifuge as above.

Repeat CaCl2 wash as above.

Decant supernatant. Add 0.6 mL 0.125 M CaCl2 and 0.4 mL 50% glycerol and gently resuspend by repipetting. Add cells to pre-cooled microcentrifuge tubes in 55 uL aliquots. Store at -80C.

Transformation

Pre-heat heat block to 42C. Fill wells that you will use with DI water. Fill an ice bucket with ice.

Put CC cells on ice and allow to thaw for ~2 min. Add 1 uL 40% PEG (in 4C) to each 55 uL aliquot of CC cells.

Add 0.5-3 uL plasmid (0.5 uL if they are miniprepped - supercoiled DNA increases transformation efficiency. If plasmid is a ligation product, use 3 uL - this plasmid is not supercoiled, which reduces the efficiency. If the plasmid is a ligation product of a digested plasmid and annealed oligos, the plasmid is nicked, which further reduces transformation efficiency).

Put on ice 30 min.

Put on heat block (in filled wells)1 min, 10 s.

Put back on ice for 2 min.

Add 400 uL SOC.

Put in 37C incubator for 1 hr.

Continue protocol @Plating(see below).