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Team:NTNU Trondheim/Protocols
From 2012.igem.org
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*45 s heat shock in stead of 60 s. | *45 s heat shock in stead of 60 s. | ||
*LB medium in stead of SOC. | *LB medium in stead of SOC. | ||
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| + | Supercompetent E. coli DH5a | ||
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==Inoculation after transformation== | ==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 and incuate at 37 C with shaking. | + | 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. |
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| + | ==DNA Isolation== | ||
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| + | We use the Promega Wizard Plus SV Minipreps DNA Purification System A1460 with the sentrifugation version of the [[http://www.promega.com/resources/protocols/technical-bulletins/0/wizard-plus-sv-minipreps-dna-purification-system-protocol/ protocol] supplied by the vendor. | ||
==Recipes== | ==Recipes== | ||
Revision as of 09:26, 22 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 [[http://www.promega.com/resources/protocols/technical-bulletins/0/wizard-plus-sv-minipreps-dna-purification-system-protocol/ protocol] supplied by the vendor.
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: 100 mg/mL dissolved in 20 w/v % 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: 25 ug/mL = 0.25 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.
