Team:UGA-Georgia/Protocols

PCR

1. Amplified mCherry gene with negative control group and positive control group
2. Reaction Mixture (a master mix to be divided into 3 tubes; Mq water 10.2 uL X 4 = 40.8 uL, 2x Phusion polymerase HF buffer mix 12.5 uL x 4 = 50 uL, Gene 0.5 uL x 4 = 2 uL, Forward primer 0.9 uL X 4= 3.6 uL, Reverse primer 0.9 uL x 4 = 3.6 uL)
3. Final Reaction Mixture = 96.4 uL with 24.1 uL in each tube
4. Annealing temperature = 65 Celsius
5. Run products on a gel for verification (2.5 uL of each)

Plasmid Extraction

1. Add 1000 uL of culture to each centrifuge tube
2. Centrifuge tubes at 15000 rpm for 30 seconds
3. Dispose supernatant in biohazard bin
4. Add another 1000 uL of each culture to respective centrifuge tubes
5. Centrifuge again at 15000 rpm for 30 seconds
6. Discard supernatant in biohazard bin
7. Add 600 uL of DI water to each centrifuge tube
8. Vortex tubes to resuspend the pellet into the water
9. Add 100 uL of 7x lysis buffer to each tube and mix for 30 seconds
10. Add 350 uL of neutralization buffer and mix
11. Centrifuge at 15000 rpm for 3 minutes
12. Transfer supernatant to spin column
13. Centrifuge at 15000 rpm for 30 seconds
14. Discard liquid in collection tube
15. Add 200 uL of endo wash buffer to each spin column
16. Centrifuge at 15000 rpm for 30 seconds
17. Add 400 uL of zippy wash buffer to each spin column
18. Centrifuge at 15000 rpm for 1 minute
19. Place spin columns in centrifuge tubes
20. Add 40 uL of DI water to each column
21. Let stand for 2 minutes (to allow the gene and plasmid to dissolve in the water)
22. Centrifuge at 15000 rpm for 30 seconds
23. Store at -20 Celsius
24. Dispose of spin columns