User:Matthewmeisel/Notebook/2006-6-20

Transformation of standard components
 * cultured bacteria did grow overnight
 * 1 mL of 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 decanted into several 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).
 * 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) Combined the supernatants from step 4 in a single 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 to verfiy that the insert was inserted
 * 1) The following ingredients were each added to a 1.5 ml centrifuge tube:
 * 2) *10 &mu;l water
 * 3) *8 &mu;l DNA from the previous step
 * 4) *2.5 &mu;l 10x NEB buffer (#2)
 * 5) *2.5 &mu;l 10x BSE
 * 6) *1.0 &mu;l 1:1 diluted enzyme A (SpeI)
 * 7) *1.0 &mu;l 1:1 diluted enzyme B (XbaI).
 * 8) Tubes were incubated at 37&deg;C for 3 h.
 * 9) Tubes were incubated at 80&deg;C for 15 min in order to inactivate the enzymes.
 * 10) 10 of digest reaction and 10  water were mixed and run in a 1.2% agarose Invitrogen E-gel for 30 min. (ladder in lane 1, our sample in lane 4).