Difference between revisions of "Team:CCA San Diego/protocols"

• DNA miniprrep
• LB agar plates, Cat No. Teknova, appropriate antibiotic
• DH5a competent cells, Invitrogen, Cat 18265-017
• SOC (Recovery Medium), Lucigen, Cat No. F98226
• 1.5 mL tube
• Vortex
• Pipet and tips
• Ice bucket and ice
• Water bath (42°C)
• Incubator (37°C) and shaker

1. Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
2. For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
3. Turn on incubator-shaker at 37°C.
4. Turn on incubator for plates at 37°C.
5. Set up water bath at 42°C.
6. Bring to room temperature S.O.C medium.
7. Bring LB plates supplemented with appropriate antibiotic at room temperature.
8. Thaw competent cells on ice.
9. Aliquots competent cells in as many tubes as needed.
10. Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
11. Incubate on ice for 20 minutes
12. Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
13. Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube
14. Incubate in shaker at 37°C, 225 rpm for 30 min
15. Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)
16. Incubate plates at 37°C overnight
17. Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion