Inhibitor expression
Introduction
Materials
- Plate of transformed E.coli BL21(DE3)
- Cultivation tubes of 10mL sterilized LB medium
- 100mg/mL ampicillin stock
Procedure
- Add ampicillin stock to cultivation tubes of 10mL sterilized LB medium
- Incolulate transformed E.coli BL21 in 10mL LB media with antibiotic using an inoculation loop and incubate at 37°C with shaking (170 rpm) overnight.
- Sample ON cultures and measure OD at 600 nm. Calculate amount needed to be added in 0.5mL of OD = 3
- Mix TB components in 3 shake flasks per clone and 0.1mL add ampicillin stock, final concentration 100µg/mL
- Innoculate shake flasks containing 100mL TB media with volume of culture prepared in step 2. Also inoculate 1 shake flask with
- Take 100µL sample from one selected tracking flask per clone and do 10x dilution in cuvette to measure OD at 600 nm
Cuvettes can be prepared ahead of time with 900µL dH2O
Don’t forget to blank spectrophotometer! If diluting: blank with dH2O, if not diluting: blank with TB
- Incubate at 37°C for 3-4 hours at 170rpm
- Prepare SDS-PAGE buffer and IPTG solution
- After 1.5-2h start making OD measurements every 30 minutes: Take 100µL sample from one flask per clone, dilute 10x in cuvette and measure OD
- Induce cells when OD reaches around 0.8 according to procedure below:
- Take 100µL sample for OD, take 500µL sample for SDS-page, add 100µL 1M IPTG stock
Procedure: Take flask out of incubator, shake manually, take 100µL sample for OD , put in cuvette, shake again, take 500µL sample for SDS, put into tube
Avoid getting droplet on the pipette tip, especially for OD measurements
Mark time of induction!
For SDS page sample:
- Centrifuge SDS-PAGE sample 1min 8 000 rpm (eppendorf centrifuge) → small cell pellet, keep on ice!
- Remove the supernatant, keep on ice
- Calculate volume to resuspend in to reach OD = 10 in SDS loading dye
- resuspend with pipette
- Then do heat treatment 5 min at 100 degrees in heat block
- Put in freezer for the next day
Need to measure for each flask being induced
- 30 min after induction of the first flask of each clone: take OD and SDS-page samples from the second flask of that clone and induce that flask with 100µL IPTG stock. After 30 more minutes, repeat for the next flask.
- At 1 h, 2h and 4h after induction: take OD sample and SDS-PAGE sample from all flasks with that induction time
- Leave cultures in shake flasks ON
- First thing next morning: Take OD and SDS-PAGE samples from all flasks
Do 20x dilution for OD measurement
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