Test Expression of E.coli in flasks

Name:

Date:

Project:

Booking

Please book the equipment listed below via our online booking system PPMS or by scanning the QR code on the system itself. You don't have a PPMS account yet? Feel free to contact us. 

Outline

To Do

please ensure the following is at hand and prepared in sufficient amounts

Day 1, date:

• Overnight Culture
  • mix 6mL LB with 6µL of your desired selection marker in a 15mL falcon tube
  • pick single colony from fresh streak transformation on LB-Agar plate
  • transfer into 6mL LB and close the falcon tube
  • growth at ______ °C, ______ RPM over night (e.g. 37°C, 180 RPM)

Day 2, date:

• Testexpression Culture
  • add 100µL desired selection marker to 100mL growth medium of choice in 250mL flask
  • inoculate with ______ mL overnight culture (e.g. 2-5mL)
  • measure starting OD₆₀₀
  • follow cell growth at ______ °C, ______ RPM (e.g. 37°C, 180 RPM) measuring OD₆₀₀ until
    
    • LB media: OD₆₀₀ = 0.6
    • Auto Inducing Media: OD₆₀₀ = 0.6
    • TB media: OD₆₀₀ = 1.2

^{table 01 - time / OD₆₀₀ until t0}

• take 1mL sample for SDS PAGE/Western Blot before induction (t0)
  • spin down 1mL sample in 1.5mL Eppi at fullspeed in tabletop centrifuge
  • discard supernatantm make sure no liquid remains in the Eppi
  • freeze pellet in liquid nitrogen, annote OD₆₀₀ to every frozen sample
  • keep at -20°C until further use

• Induction
  • LB/TB media: induce expression with ______ mM inducing reagent (IPTG, AHT, arabinose) (e.g. 0.1mM - 1mM)
  • Auto Inducing Media: switch temperature from ______ °C (e.g. 37°C) to ______ °C (e.g. 20°C or 30°C)

• measure OD₆₀₀ each 1h after induction
• take 1mL samples each 1h, up to 5h after induction (t1, t2, etc)
  • proceed with 1mL samples as described above

• annotate OD₆₀₀ to specific timepoints in table 02

^{table 02 - measuring points after induction with different temperatures}

• eventually keep cultures growing overnigh

Day 3, date:

• Test Expression Culture and SDS PAGE and Western Blot
  • before ending the expression experiment, measure OD600 and take another 1mL sample for SDS PAGE and Western Blot analysis
    • proceed with 1mL samples as described above

^{table 03 - OD₆₀₀ after overnight growth}

• depending on the number of samples you need 1-2 SDS PAGE for analysis
  • 1x SDS PAGE
  • 1x Western Blot

• Loading the Gel
  • please ensure to only take one sample from -20°C at a time, to avoid a sticky mess while
  • prepare 5µL of 4x SDS Loading Dye / Sample in 1.5mL eppi
  • take out sample from -20°C, briefly warm up in hands
  • dilute sample with ddH₂O
    • 100µL ddH₂O/OD₆₀₀=0.1
    • e.g. OD₆₀₀=0.6, dilute pellet with 600µL ddH₂O
  • immediately transfer 20µL of resuspended cell pellet to 5µL 4x SDS Loading Dye and mix well
  • heat sample to __________ °C for __________ min. (optional)
  • load __________ µL of prepared sample into SDS PAGE gel (depending on the wellcomb used, 7-10µL)
    • if you are using two gels, please ensure to load both gels in the same order for better compairson of SDS PAGE and Western Blot
  • continue with all remaining samples as described above
  • use PreStained Marker for Western Blot

• annotate your sample sequence here

• run SDS PAGE and Western Blot referring to our standard protocols (SDS PAGE: constant 120V, 90min. Western Blot: 25V, 1.0A for 30min (BioRad TransBlot Turbo))

Results and Conclusions