User:Matthewmeisel/Notebook/2006-6-20: Difference between revisions
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Revision as of 13:39, 10 July 2006
Week 2
Tue Jun 20
Transformation of standard components
- cultured bacteria did grow overnight
- 1 mL of reserved to make glycerol stock:
- 666 μL of cells and medium and 666 μL of 50% glycerol solution in water
- stored at 80[[:Category:{{{1}}}|{{{1}}}]]
- extracted DNA from remaining 4 mL using the standard protocol:
- Cells and medium decanted into several 1.5 mL microcentrifuge tubes, centrifuged, and supernatant discarded.
- Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C).
- Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
- Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.
- Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.
- Combined the supernatants from step 4 in a single QIAprep spin column by decanting.
- Centrifuged for 60 s. Discarded the flow-through.
- Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.
- Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
- 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
- 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).
- Tubes were incubated at 37°C for 3 h.
- Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
- 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).