Page tree

Versions Compared

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.

...

name: __________________, date: ________________

Materials

...

LB Media / 1L

...

ZY Media 928mL/L for auto-inducing

...

20x NPS 50mL/L

...

50x 5052 20mL/L

...

2mM MgSO4

...

10g Peptone

...

10g Peptone

...

1M Na2HPO4

...

25% Glycerol

...

1M MgSO4

...

5g Yeastextract

...

5g Yeastextract

...

1M KH2PO4

...

2.5% Glucose

...

10g NaCl

...

0.5M (NH4)2SO4

...

10% a-Lactose

...

table 01 - components for LB or Auto Inducing Media

- 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

...

glass

...

plastic

...

baffled

...

unbaffled

...

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)

...

Time

...

OD600

...

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)

...

Time

...

OD600

...

Mainculture Auto Inducing Media
-
bevore harvesting check OD600

...

Time

...

OD600

...

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

...