Let the gel run for 45 min at 90 V, and if the bands are porly separated, run a little longer. Beware that if the gel runs for a very long time, the smaller bands may run out of the gel.
Let the gel run for 45 min at 90 V, and if the bands are porly separated, run a little longer. Beware that if the gel runs for a very long time, the smaller bands may run out of the gel.
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.
Add 2 uL of each sample to be ligated (insert and backbone)
Add 2ul of T4 DNA Ligase Reaction Buffer
Add 1ul of T4 DNA Ligase
Mix well, and spin down
Incubate for 30min at 16C and 20min at 80C to heat kill
Use 2ul of ligation to transform into competent cells
In stead of incubating as described above, the samples are kept at 37 degrees celsius in a water bath for 60 minutes.
Gel purification
We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com.
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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.