From 2014.igem.org
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PCR |
- Amplified mCherry gene with negative control group and positive control group
- 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)
- Final Reaction Mixture = 96.4 uL with 24.1 uL in each tube
- Annealing temperature = 65 Celsius
- Run products on a gel for verification (2.5 uL of each)
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Plasmid Extraction |
- Add 1000 uL of culture to each centrifuge tube
- Centrifuge tubes at 15000 rpm for 30 seconds
- Dispose supernatant in biohazard bin
- Add another 1000 uL of each culture to respective centrifuge tubes
- Centrifuge again at 15000 rpm for 30 seconds
- Discard supernatant in biohazard bin
- Add 600 uL of DI water to each centrifuge tube
- Vortex tubes to resuspend the pellet into the water
- Add 100 uL of 7x lysis buffer to each tube and mix for 30 seconds
- Add 350 uL of neutralization buffer and mix
- Centrifuge at 15000 rpm for 3 minutes
- Transfer supernatant to spin column
- Centrifuge at 15000 rpm for 30 seconds
- Discard liquid in collection tube
- Add 200 uL of endo wash buffer to each spin column
- Centrifuge at 15000 rpm for 30 seconds
- Add 400 uL of zippy wash buffer to each spin column
- Centrifuge at 15000 rpm for 1 minute
- Place spin columns in centrifuge tubes
- Add 40 uL of DI water to each column
- Let stand for 2 minutes (to allow the gene and plasmid to dissolve in the water)
- Centrifuge at 15000 rpm for 30 seconds
- Store at -20 Celsius
- Dispose of spin columns
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Gel Extraction |
- Add 2 uL of DNA ladder and 50 uL of digested vector with 10 uL loading buffer dye into gel
- Run gel at 100 Volts until markers move half way down the gel
- Soak Gel in Ethidium Bromide for 10 minutes
- Observe gel under UV light
- Carefully cut out the part of gel which contain the vector and put into the centrifuge tube
- Add 200 uL of ADB buffer into each centrifuge tubes
- Place the centrifuge tubes in a 55 Celsius water bath for 20 minutes
- Shake well and place them back in water for 5 more minutes
- Pipet the solution from each tube into spin column-collection tubes
- Centrifuge at 8000 rpm for 1 minutes, and discard the supernatant
- Add 200 uL of DNA wash buffer to each spin basket
- Wait 2 minutes; then centrifuge at 15000 rpm for 30 seconds and discard the supernatant
- Transfer spin baskets to centrifuge tubes, and add 8 uL of water into each tube
- Let it sit for 2 minutes
- Centrifuge at 15000 rpm for 30 seconds
- Collect purified gene into one centrifuged tube
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