This is a list of recipes and protocols used by the team.
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Official iGEM transformation protocol: http://partsregistry.org/Help:Protocols/Transformation
We use this protocol with the following modifications:
The cells used are Subcloning Efficiency™ DH5α™ Competent Cells from Life Technologies/Invitrogen
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.
We use the Promega Wizard Plus SV Minipreps DNA Purification System A1460 with the sentrifugation version of the protocol supplied by the vendor.
Concentrations of DNA after isolation was measured with the NanoDrop ND-1000 Spectrophotometer
We are using the single reaction protocol from partsregistry.org : [1]
Ligation protocol from partsregistry.org [2]:
In addition, the samples are incubated at 37 degrees celsius in a water bath after spinning down.
We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com. [3]
LB-medium (LB-Lennox):
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.
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