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

Restriction Digestion

1. PCR purified product, plasmid to be digested separately - choose the restriction enzymes and the buffer
2. Set up: 24 uL of water, 5 uL of 10X buffer, 5 uL of 10X BSA, 2 uL of Restriction Enzyme 1, 2 uL of Restriction Enzyme 2, 12 uL of Vector/DNA
3. Incubate at 37 Celsius for 2-4 hours in water bath

Gel Extraction

1. Add 2 uL of DNA ladder and 50 uL of digested vector with 10 uL loading buffer dye into gel
2. Run gel at 100 Volts until markers move half way down the gel
3. Soak Gel in Ethidium Bromide for 10 minutes
4. Observe gel under UV light
5. Carefully cut out the part of gel which contain the vector and put into the centrifuge tube
6. Add 200 uL of ADB buffer into each centrifuge tubes
7. Place the centrifuge tubes in a 55 Celsius water bath for 20 minutes
8. Shake well and place them back in water for 5 more minutes
9. Pipet the solution from each tube into spin column-collection tubes
10. Centrifuge at 8000 rpm for 1 minutes, and discard the supernatant
11. Add 200 uL of DNA wash buffer to each spin basket
12. Wait 2 minutes; then centrifuge at 15000 rpm for 30 seconds and discard the supernatant
13. Transfer spin baskets to centrifuge tubes, and add 8 uL of water into each tube
14. Let it sit for 2 minutes
15. Centrifuge at 15000 rpm for 30 seconds
16. Collect purified gene into one centrifuged tube

Ligation

1. Made a solution of 60 uL of 2x buffer, 12 uL of digested vector, and 6 uL of quick ligase
2. For each of the new centrifuge tubes(12 in total, properly labeled), add 3.5 uL of the previous PCR product and 6.5 uL of the solution into each tube in the order as labeled
3. Let it sit for 10 minutes

Heat Shock Transformation

1. Add 10 uL of the plasmid from ligation to each centrifuge tube, which contain 50uL of XL1-Blue cells, in the order as labeled
2. Keep on ice for 30 minutes
3. Heat shock at 42 Celsius for 60 seconds (6 tubes each time)
4. Add LB, and keep on ice in 37 Celsius shaker for 45 minutes
5. Centrifuge at 5000 rpm for 3 minutes
6. Discard 600 uL supernatant
7. Re-suspend the 100 uL culture
8. Plate the 100 uL on each LB-ampicillin plate as labeled
9. Place in the 37 Celsius Incubator