User:Matthewmeisel/Notebook/2006-6-14

Transformation of standard components
 * cultured bacteria did grow overnight
 * 1 mL of each tube reserved to make glycerol stock:
 * 1) 666  of cells and medium and 666  of 50% glycerol solution in water
 * 2) stored at 80
 * extracted DNA from remaining 4 mL using the standard protocol:
 * 1) Cells and medium poured into 1.5 mL microcentrifuge tubes, centrifuged, and supernatant discarded.
 * 2) Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 &deg;C) and transfered to a microcentrifuge tube.
 * 3) Added 250 &mu;l Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
 * 4) Added 350 &mu;l Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.
 * 5) Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.
 * 6) Applied the supernatants from step 4 to the QIAprep spin column by decanting.
 * 7) Centrifuged for 60 s. Discarded the flow-through.
 * 8) Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.
 * 9) Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
 * 10) Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 &mu;l water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.
 * digested promoter (R0010) and GFP (E0241) plasmids
 * R0010 digested with SpeI and PstI in order to leave it attached at upstream end to the plasmid backbone (otherwise, the fragment would only be ~200 bp long, which is a little too short for electrophoresis with much longer fragments
 * E0241 digested with XbaI and PstI in order to cleave it as a fragment
 * 1) The following ingredients were each added to four 1.5 ml centrifuge tubes:
 * 2) *10 &mu;l water
 * 3) *8 &mu;l DNA from the previous step (R0010-1, R0010-2, E0241-1, E0241-2 in respective tubes)
 * 4) *2.5 &mu;l 10x NEB buffer (#2 for R0010-01 and R0010-2, #3 for E0241-1 and E0241-2)
 * 5) *2.5 &mu;l 10x BSE
 * 6) *1.0 &mu;l 1:1 diluted enzyme A (SpeI for R0010-1 and R0010-2, XbaI for E0241-1 and E0241-2)
 * 7) *1.0 &mu;l 1:1 diluted enzyme B (PstI for all).
 * 8) Tubes were incubated at 37&deg;C for 90 min.
 * 9) Tubes were incubated at 80&deg;C for 15 min in order to inactivate the enzymes.
 * 10) All four samples were run on a 1% agarose gel: