User:Matthewmeisel/Notebook/2006-6-20

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Transformation of standard components

  • cultured bacteria did grow overnight
  • 1 mL of 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 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 °C).
  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. 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 μl 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:
    • 10 μl water
    • 8 μl DNA from the previous step
    • 2.5 μl 10x NEB buffer (#2)
    • 2.5 μl 10x BSE
    • 1.0 μl 1:1 diluted enzyme A (SpeI)
    • 1.0 μl 1:1 diluted enzyme B (XbaI).
  2. Tubes were incubated at 37°C for 3 h.
  3. Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
  4. 10 μL of digest reaction and 10 μL 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).
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