User:Matthewmeisel/Notebook/2006-6-15

Transformation of standard components
 * ligation and transformation of promoter and GFP
 * ligation protocol with the Roche Rapid DNA ligation kit:
 * 1) 6  of insert (E0241), 1  of plasmid (R0010 and backbone), and 3  of 1x DNA dilution buffer (vial 2) (enough buffer for total volume of 10 ) into a microcentrifuge tube. (Would ordinarily use 2  of plasmid, but the plasmid concentration is about doubled because it combined bands from two gel lanes.)
 * 2) Mixed ligation buffer (vial 1), and added 10  to tube.
 * 3) Added 1  T4 DNA ligase (vial 3).
 * 4) Incubated on ice for 5 min.
 * transformation:
 * 1) 30  competent cells each into three microcentrifuge tubes.
 * 2) Ligation mixture, 1  water (negative control), and 1  pUC 19 (positive control) added to respective tubes.
 * 3) Incubated the tubes on ice for 20 min.
 * 4) Heat shocked the tubes at 42&deg;C for 30 s.
 * 5) Added 200  SOC to each tube.
 * 6) Incubated, with shaking, at 37&deg;C for 1 h.
 * 7) Plated the mixture on carbomycin plates and incubated at 37&deg;C for 24 h, then stored at 4&deg;C.