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name: __________________, date: ________________


Materials

LB Media / 1L

Auto Inducing Media 928mL/L

20x NPS 50mL/L

50x 5052 20mL/L

2mM MgSO4

10g Peptone

10g Peptone

1M Na2HPO4

25% Glycerol

2mL from 1M MgSO4

5g Yeastextract

5g Yeastextract

1M KH2PO4

2.5% Glucose


10g NaCl


0.5M (NH4)2SO4

10% a-Lactose



- LB-Media will be autoclaved in the flasks
- Auto Inducing Media (AIM) will be autoclaved without the additional ingredients. They will be sterile filtred
  only
- fresh transformation of plasmid with expression host of interest on LB-Agar with desired selection marker
  or glycerol stock

 ________________________________________________________________________________________ (e.g. BL21(DE3)::plasmid::gene)

- 2x 100mL LB in 250mL flasks for overnight culture

LB Media: - 6 – 12x 0.5L in 2L flasks, ____________________
                - 1M IPTG, ____________________ mL
                - 3 – 6mL selection marker: ____________________

AIM: - 6 – 12x 464mL in 2L flasks, ____________________
        - all other ingredients will be added before inoculating with overnight culture
        - 3 – 6mL selection marker: _____________________

glass


plastic


baffled


unbaffled



- 1x preweight 50mL Falcon Tube per 2L cell pellet, __________ mL 1x PBS - ice cold, liquid Nitrogen in dewer

Equipment
Avanti JXN-26 #2, 1000mL bottles  for cell harvesting
S1 Eppendorf 5920R for cell washing with PBS
Incubator

Innova S441 Top

Innova S441 Mid

Innova S441 Bottom

Innova 4400 Top

Innova 4400 Bottom






Preparations: - please ensure all materials needed are available in sufficient amounts
                     - please book all equipment needed online in PPMS (webinterface or QR codes)



Day 1, date: _______________
Overnight culture
- pick single colony from transformation on LB-Agar plate, date ____________________
- or scrape some small amount from glycerol stock, date ____________________
- transfer into 100mL LB supplemented with selection marker each
- growth at 37°C, __________ RPM (e.g. 180) overnight in Incubator

Day 2, date: ________________
Mainculture LB
- add selection marker to 500mL LB in 2L flasks
- measure OD600 of Overnight culture (1:10 dilution): __________
- inoculate with overnight culture to an OD600 = 0.1 (C1xV1 / C2xV2)
  OD600 = 0.1 x 500mL / OD600 = __________ x V2
  V2=__________
- ________________________________________________________________________________________________________________________
- growth at __________°C and __________ RPM in Incubator until OD600 = __________ (e.g. 37°C, 180 RPM, OD600=0.6)

Time

OD600













- take 1mL sample for SDS PAGE analysis, freeze in -20°C
- induce protein expression with __________ mM IPTG final concentration (e.g. 0.1 – 1mM)
- continue growth for __________ h at __________ °C and __________ RPM, overnight possible (e.g. 4h, 30°C, 180 RPM)
- _________________________________________________________________________________________________________________________


Mainculture Auto Inducing Media
- add selection marker to 464mL media in 2L flasks
- add 25mL/464mL 20x NPS
- add 10mL/464mL 50x 5052
- add 1mL/464mL 1M MgSO4
-
measure OD600 of Overnight culture (1:10 dilution): __________
- inoculate with overnight culture to an OD600 = 0.1 (C1xV1 / C2xV2)
  OD600=0.1 x 500mL / OD600=__________x V2
  V2=__________
- __________________________________________________________________________________________________________________________
- growth at __________ °C and __________ RPM in incubator until OD600 = __________ (e.g. 37°C, 180 PM, OD600=0.8)

Time

OD600













- take 1mL sample for SDS PAGE analysis, freeze in -20°C
- induce protein expression by setting temperatur to 30°C/20°C
- continue growth for __________ h at __________ °C and __________ RPM overnight (e.g. 16h, 20°C, 180 RPM)

- ___________________________________________________________________________________________________________________________

Day 2/3, date:  ________________
Mainculture LB
- before harvesting check OD600

Time

OD600



- take 1mL sample for SDS PAGE analysis, freeze in -20°C

Mainculture Auto Inducing Media
-
bevore harvesting check OD600

Time

OD600



- take 1mL sample for SDS PAGE analysis, freeze in -20°C

Harvesting

- transfer expression culture into 1000mL bottles for Avanti JXN-26 #2
- balance tubes correctly
- harvest cells at __________ xg for __________ min at __________ °C (e.g. 5000xg, 15min, 4°C)
- weigh 50mL falcon tubes to calculate wet cell mass


1

2

3

4

50mL falcon tube empty





------“------       With cells





Wet Cell Mass





please note: supernatants will be kept in expression flasks for autoclaving, do not put them into the sink
- discard supernatant and scrape out cell pellet with soft spatula
- transfer two cell pellets into one 50mL falcon tube
- add 30mL icecold 1x PBS per 50mL falcon tube and resuspend by pipetting
- pellet cells again by centrifuging at 3234xg  for 30min at 4°C
- disard supernatant and freeze pellets with liquid Nitrogen and keep at -80°C unti further use


Results

Liters medium


Amount cells


Amount cells per Liter


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