User:Matthewmeisel/Notebook/2006-6-20

Week 2

Tue Jun 20

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}}}]]

• 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 °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).