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Team:NTNU Trondheim/Protocols
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Ampicillin stock solutions: 100 mg/mL dissolved in MQ water<br> | Ampicillin stock solutions: 100 mg/mL dissolved in MQ water<br> | ||
Kanamycin stock solutions: 100 mg/mL dissolved in MQ water<br> | Kanamycin stock solutions: 100 mg/mL dissolved in MQ water<br> | ||
| - | Chloramphenicol: | + | Chloramphenicol: 34 mg/mL dissolved in 100% ethanol |
Store at -20 C after preparation and between use. | Store at -20 C after preparation and between use. | ||
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Ampicillin media concentration: 100 ug/mL = 1 mL stock solution/L medium<br> | Ampicillin media concentration: 100 ug/mL = 1 mL stock solution/L medium<br> | ||
Kanamycin media concentration: 100 ug/mL = 1 mL stock solution/L medium | Kanamycin media concentration: 100 ug/mL = 1 mL stock solution/L medium | ||
| - | Chloramphenicol media concentration: | + | Chloramphenicol media concentration: 24 ug/mL = 0.7 mL stock solution/L medium |
When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant. | When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant. | ||
Revision as of 14:13, 26 June 2012
This is a list of recipes and protocols used by the team.
Contents |
Transformation
Official iGEM transformation protocol: http://partsregistry.org/Help:Protocols/Transformation
We use this protocol with the following modifications:
- 45 s heat shock in stead of 60 s.
- LB medium in stead of SOC.
Supercompetent E. coli DH5a
Inoculation after transformation
Using a sterile toothpick, pick/scratch a single colony from the transformants. Drop the toothpick into a plastic tube with 3 mL sterile liquid medium with the appropriate antibiotic(s). Close the tube but leave the cap slightly open to allow oxygen to enter, and incuate at 37 C with shaking.
DNA Isolation
We use the Promega Wizard Plus SV Minipreps DNA Purification System A1460 with the sentrifugation version of the protocol supplied by the vendor.
DNA Concentration measurements
Concentrations of DNA after isolation was measured with the NanoDrop ND-1000 Spectrophotometer
Recipes
Growth media
LB-medium (LB-Lennox):
Antibiotics
Ampicillin stock solutions: 100 mg/mL dissolved in MQ water
Kanamycin stock solutions: 100 mg/mL dissolved in MQ water
Chloramphenicol: 34 mg/mL dissolved in 100% ethanol
Store at -20 C after preparation and between use.
Ampicillin media concentration: 100 ug/mL = 1 mL stock solution/L medium
Kanamycin media concentration: 100 ug/mL = 1 mL stock solution/L medium
Chloramphenicol media concentration: 24 ug/mL = 0.7 mL stock solution/L medium
When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant.
