Protocol

Abbreviation

UTSW_standard expression

Name

UTSW_standard expression

Laboratory name

University of Texas Southwestern Medical Center

Type

expression

Description

Cloning

Transformation Protocol:

Transformation of clone into 2 competent cells
1) One shot Top 10 competent cells
2) Rosetta (DE3) pLysS competent cells

Thaw the competent cells (50ml) on ice.
Warm the SOC medium in 37ºC
Add 5µL of plasmid DNA to each competent cells directly (Do not pipette, just mix by tapping)
Incubate the vial on ice for 30mins.
Incubate for exactly 30secs in 42ºC water bath (Do not shake or mix)
Remove vials from water bath and quickly place on ice.
Add 250 µL of prewarmed SOC medium to each vial.
Incubate vials at 37ºC for 1 hr at 225 rpm in shaking incubator
Plate 20-200 ml of each transformation reaction onto 3LB plates with appropriate antibiotic.
Top 10 onto LB Agar plates containing Amphicillin antibiotic
Rosetta onto LB Agar Plates containing Amphicillin + Chloramphenicol.
Incubate the plates inverted at 37ºC O/N.

Transformation efficiency is more in Top10 compared to Rosetta.

Expression:

Small LB culture:
1. To 50mL of LB media add 50 µL of 200mg/ml ampicillin ( final concentration is 200 µg/ml) – Top 10 and 50 µL of Chloramphenicol - Rosetta
2. Inoculate with single transformed colony respectively
3. Shake at 150rpm, 37ºC, O/N.

Large TB culture:
1. To 2L of TB add respective antibiotic according to the competent cells, and add 2ml of small culture into 2L of media respectively. Split to two 1L flasks
2. Shake at 250rpm, 37ºC and take samples every hour to measure OD600nm.
3. When the OD600nm is ~0.6, decrease the growing temperature to 20ºC and induce the protein expression by addition of 1ml of 1M IPTG (final concentration 1mM)
4. Grow O/N at 20ºC, 250rpm.
Expression was more in Rosetta compared to Top10.
So further purification steps are with only Rosetta.