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From 2012.igem.org

This is a list of recipes and protocols used by the team.

Contents 1 Transformation 2 Inoculation after transformation 3 DNA Isolation 4 Recipes 4.1 Growth media 4.2 Antibiotics

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.