User:Matthewmeisel/Notebook/2006-6-14

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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}}}]]
  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
01kb DNA ladder (10 μL)10x loading dye (1.1 μL)
1E0241-1 (15 μL)10x loading dye (2.5 μL)
2E0241-2 (15 μL)10x loading dye (2.5 μL)
3R0010-1 (15 μL)10x loading dye (2.5 μL)
4R0010-2 (15 μL)10x loading dye (2.5 μL)
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