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.
We are using the single reaction protocol from partsregistry.org : [1]
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.
"
