PROTOCOL FOR E. COLI TRANSFORMATION
Stock Solutions:
1. Ampicillin (100 mg/ml): add 1 g ampicillin to 10 ml dH2O, sterilize solution through syringe
filter (0.2 mM), prepare 1.0 mL aliquots in individual eppendorf tubes, and store at –20º C.
2. Kanamycin (100 mg/ml): add 1 g kanamycin to 10 ml dH2O, sterilize solution through syringe
filter (0.2 mM), deliver 1 mL aliquots into individual eppendorf tubes, and store at -20.
3. 80% glycerol: mix 80 ml glycerol and 20 ml dH2O in Wheaton bottle (size 250 mL) and
autoclave.
4. 0.1 M IPTG: 0.0238 g IPTG + 1 mL dH2O.
5. LB-amp Broth: add 2.5 g LB-broth powder to 100 mL dH2O, mix by swirling to dissolve
powder, and autoclave. Add 100 µL ampicillin stock to 100 mL LB-broth solution at room
temperature after autoclaving.
6. LB-amp Agar: add 3.7 g LB-agar powder to 100 mL dH2O, mix by swirling to dissolve
powder, and autoclave. Add 100 µL ampicillin stock to 100 mL LB-agar solution shortly after
autoclaving when solution has cooled to 50º C, mix thoroughly and let harden for 1-2 hours.
7. Plasmids: PTREC2 (encodes residues 1-202 of bovine recoverin) and pBB131-NMT.
Transformation of PTREC2 and PBB131 Plasmid DNA into E. coli Cells
1. Prepare LB Agar plates: add 3.7 g LB Agar into 100 mL H2O, autoclave, cool agar to 42º C, add
antibiotics (ampicillin (100 mg/L) and kanamycin (100 mg/L)), pour liquid agar into 4 plates, and
let agar harden for 1-2 hours.
2. Thaw competent cells (DH5α) slowly on ice.
3. Add 50 µL competent cells (DH5α) + 1 µL (0.1 µg) of PTREC2 + 1 µL (0.1 µg) PBB131 plasmids
into sterile eppendorf tube and incubate on ice for 20 min.
4. Place cells in water bath and rapidly raise temperature to 42° C for 30 seconds (water bath).
5. Cool cells on ice for 2 min.
6. Add 50 mL transformation mixture into 0.9 mL LB broth in sterile culture tube (5 mL size).
7. Grow cells with shaking (225 rpm) for 2 hours at 30° C.
8. Spread 100 µL of cells onto LB agar plate (previously prepared and containing ampicillin and
kanamycin) and let cells grow 16-24 hours at 30° C.
9. Identify single colonies on the plate to be used for producing frozen stock culture.
Frozen Stock Cultures of Transformed Cells.
1. Pick a single colony from plate above using sterilized loop and inoculate 2 mL LB broth
(containing ampicillin (100 mg/L) and kanmycin (100 mg/mL)) in sterile culture tube.
2. Grow culture with shaking (250 rpm) 3-4 hours or overnight at 30° C (until A600 = 1.0).
3. Add 1% inoculum and 10 mL LB Broth (containing ampicillin (100 mg/L) and kanmycin (100
mg/mL)) in sterile 50 mL Falcon tube.
4. Grow culture (10 mL) with shaking (250 rpm) for 3-4 hours or overnight at 30° C (until A600 = 12).
5. Add 1 mL aliquot of culture to 0.25 mL 80% glycerol into sterile, sealed cryogenic tube (makes 10
stock cultures).
6. Flash freeze the sealed tubes in liquid N2 and store at –70° C.
Screening Cells for Protein Expression.
1. Thaw frozen cells from above and add 500 µl of cell mixture to 3 mL of LB-amp/kan in sterile
culture tube and grow cells with shaking (250 rpm) 1-2 hours at 32º C (until A600 = 1-2).
2. Add 3 mL pre-culture to 30 mL LB-amp/kan broth (15 mL each in two 50mL Falcon tubes or
sterile Erlenmeyer flask) and grow cultures with shaking (250 rpm) for ~2 hours at 32º C (until
A600 = 0.5).
3. Add myristic acid (10 mg/l) and 0.5 mM IPTG to one 15 ml culture but not the other (negative
control) to induce expression of NMT and grow cells for 30 min at 32º C.
4. Add 3 mL of hot LB-medium (90 º C) to 15 mL culture and grow cells at 42º C for one hour to
induce expression of recoverin.
5. Harvest cells by centrifugation (4000 rpm for 10 min.), discard supernatant, suspend pellet in 1mL
of lysis buffer, and sonicate (power = 3, 50% duty cycle for 30 sec) to lyse cells.
6. Save 15 µL aliquot of sonicate, spin down cell debris and save 15 µL aliquot of supernatant. Treat
both aliquots with equal volume of SDS-PAGE treatment buffer and run both samples on SDSPAGE.