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 μL of cells and medium and 666 μL of 50% glycerol solution in water
2. stored at 80[[:Category:{{{1}}}|{{{1}}}]]

• 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 °C) and transfered to a microcentrifuge tube.
3. Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
4. Added 350 μl 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 μl 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:
   • 10 μl water
   • 8 μl DNA from the previous step (R0010-1, R0010-2, E0241-1, E0241-2 in respective tubes)
   • 2.5 μl 10x NEB buffer (#2 for R0010-01 and R0010-2, #3 for E0241-1 and E0241-2)
   • 2.5 μl 10x BSE
   • 1.0 μl 1:1 diluted enzyme A (SpeI for R0010-1 and R0010-2, XbaI for E0241-1 and E0241-2)
   • 1.0 μl 1:1 diluted enzyme B (PstI for all).
2. Tubes were incubated at 37°C for 90 min.
3. Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
4. All four samples were run on a 1% agarose gel:

Lane	Contents	Loading Buffer
0	1kb DNA ladder (10 μL)	10x loading dye (1.1 μL)
1	E0241-1 (15 μL)	10x loading dye (2.5 μL)
2	E0241-2 (15 μL)	10x loading dye (2.5 μL)
3	R0010-1 (15 μL)	10x loading dye (2.5 μL)
4	R0010-2 (15 μL)	10x loading dye (2.5 μL)