Biology Protocols

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Autoinduction Media Protocol

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Medias required (for component recipes go here):

ZYP-0.8G (for starter cultures)

  • Rich medium for growth with little or no induction
  • Culture should go somewhat acid at saturation (slightly below pH 6)
  • Collect cultures for freezer stocks well before saturation
  • For all media, add 1 M MgSO4 before adding 20xNPS to avoid precipitate
  • Kanamycin is used at significantly higher concentrations (100 µg/ml) than is normally the case (25-40 µg/ml). Studier has found that in the T7 expression strains in these rich media, it does not provide adequate selection at the lower concentrations.

Component

50 ml

100 ml

200 ml

400 ml

Concentration

ZY

~46.5 ml

~93 ml

~186 ml

~372 ml

-

1 M MgSO4

50 µl

100 µl

0.2 ml

0.4 ml

1 mM

1000x metals mix

50 µl

100 µl

0.2 ml

0.4 ml

1 X

40% glucose

1 ml

2 ml

4 ml

8 ml

0.8%

20x NPS

2.5 ml

5 ml

10 ml

20 ml

1x

Antibiotics, as needed

Choose only 1 from the list below

Choose only 1 from the list below

Choose only 1 from the list below

Choose only 1 from the list below

 

kanamycin (25 mg/ml)

200 µl

0.4 ml

0.8 ml

1.6 ml

100 µg/ml

chloramphenicol (25 mg/ml)

50 µl

100 µl

0.2 ml

0.4 ml

25 µg/ml

ampicillin (50 mg/ml)

50 µl

100 µl

0.2 ml

0.4 ml

50 µg/ml

ZYP-5052 rich medium for auto-induction (bacterial growth and expression of proteins)

  • For all media, add 1 M MgSO4 before adding 20xNPS to avoid precipitate
  • Kanamycin is used at significantly higher concentrations (100 µg/ml) than is normally the case (25-40 µg/ml). Studier found that in the T7 expression strains in these rich media, it does not provide adequate selection at the lower concentrations.
  • Use 400 ml in a 2 liter baffled flask (if available).
    Adequate aeration is essential to the performance of this media. Don't use more than 20% of the nominal volume of the flask.
    Baffled flasks will give significantly better performance. You might obtain adequate results with non-baffled flasks, but I don't recommend it.

 

Component

200 ml

400 ml

500 ml

1 liter

Concentration

ZY

~186 ml

~372 ml

~464 ml

~928 ml

-

1 M MgSO4

0.2 ml

0.4 ml

0.5 ml

1 ml

1 mM

1000x metals mix

200 µl

400 µl

500 µl

1 ml

50x 5052

4 ml

8 ml

10 ml

20 ml

1x

20x NPS

10 ml

20 ml

25 ml

50 ml

1x

Antibiotics

Choose only 1 from the list below

 

 

 

 

kanamycin (25 mg/ml)

0.8 ml

1.6 ml

2 ml

4 ml

100 µg/ml

chloramphenicol (25 mg/ml)

0.2 ml

0.4 ml

0.5 ml

1 ml

25 µg/ml

ampicillin (50 mg/ml)

0.2 ml

0.2 ml

0.5 ml

1 ml

50 µg/ml

 

Autoinduction protocol

  • Set up starter cultures of the bacterial colonies harbouring the plasmid to be used for overexpression in 2ml ZYP-0.8G (with an appropriate antibiotic) and grow over the day at 37°C until the cultures begin to look moderately cloudy (~6-8hours).
  • Use 200µl of starter culture to inoculate 400ml of ZYP-5052 and grow cultures overnight at your chosen temperature (If growing at lower temperatures growth and expression can take significantly longer to reach endpoint).
  • In the morning (after ~16hrs) recover bacteria by centrifuging at 12,000 rpm for 12 minutes and store cell pellets at -20°C until required (Average pellet =~3.5g per 400ml culture).


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