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.
The cells used are [http://products.invitrogen.com/ivgn/product/18265017 Subcloning Efficiency™ DH5α™ Competent Cells] from Life Technologies/Invitrogen
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/~/media/files/resources/protocols/technical%20bulletins/0/wizard%20plus%20sv%20minipreps%20dna%20purification%20system%20protocol.pdf?la=en protocol] supplied by the vendor.
DNA Concentration measurements
Concentrations of DNA after isolation was measured with the [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer]
Restriction digest
We are using the single reaction protocol from partsregistry.org : [http://partsregistry.org/Help:Protocols/Restriction_Digest]
250 ng of DNA is added together with the appropriate amount of dH2O, for a total volume of 16 uL.
Add 2,5 uL of NED-buffer 2
Add 0,5 uL og BSA
Add 0,5 uL of Enzyme 1
Add 0,5 uL of Enzyme 2
The total volume should now be 20 uL. Mix well and spin down.
Incubate the restriction digest at 37C for 1h
Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
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.
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