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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