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Team:TzuChiU Formosa/Protocol
From 2009.igem.org
(Difference between revisions)
| Line 134: | Line 134: | ||
Cycle 34 times | Cycle 34 times | ||
25℃ 2 min | 25℃ 2 min | ||
| - | + | 7. The PCR product was examed by electrophoresis in 1％ agarose. | |
| Line 143: | Line 143: | ||
| - | + | #Transfer 1.5ml of bacterial culture to each well(24 wells plate) | |
| - | + | #pellet cells by centrifuging at 33000 rpm for 10min at 4℃ | |
| - | + | #carefully remove the supernatant | |
| - | + | #add 100μl of Solution 1 (*)to each well, and vortex | |
| - | + | #add 200μl of Solution 2 (*)to each well, and mix gently | |
| - | + | #add 150μl of Solution 3 (*)to each well | |
| - | + | #centrifuge 3000rpm at 4℃ for 10min | |
| - | + | #add 1 ml 100% EtOH to new well | |
| - | + | #Transfer the supernatant to the new well, containing 100% EtOH. | |
| - | + | #centrifuge 3300rpm at 4℃ for 30min. | |
| - | + | #carefully remove the supernatant. | |
| - | + | #add 75% EtOH to wash pellets, then remove the supernantant, then air dry. | |
| - | + | #add 40μl ddH2O to each well, to dissolve with plasmid DNA | |
| - | + | #store the plate in 4℃ | |
(*)Sol 1: 10m Miris/0.5mM HEDTA, PH 7.4 buffer. | (*)Sol 1: 10m Miris/0.5mM HEDTA, PH 7.4 buffer. | ||
Sol 2: 0.2N NaOH/1% SDS | Sol 2: 0.2N NaOH/1% SDS | ||
Sol 3: 3M KOAC/HOAC | Sol 3: 3M KOAC/HOAC | ||
| - | ====Digestion | + | ====Digestion==== |
Revision as of 15:47, 21 October 2009
Contents |
Protocol
A. Culture CP919
B. Put Aqueorin-GFP into plasmid .
C. Transform the cp919 into the Midnight Apollo!
D. Sense the light.
E. In the dark, the Midnight Apollo! is activated and emit the light.
Competent Cell (CP919-Cph8)
- Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).
- Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.
- Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.
- Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)
- Spin down the bacteria at 4℃,3000 rpm for 10 min.
- Discard the supernatant and mix the cell pellet with 10ml FSB.
- Keep the cells on ice for 3~4 hours.
- Spin down, at 4℃,3000 rpm for 10 min.
- Discard the supernatant and mix the cell pellet with 5ml FSB.
- Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.
Tansformation Protocol
- Take competent cell in an eppendorf tube from -80℃ freezer put in ice.
- Add 2ul plasmid to competent cell and place in ice for 5 minutes.
- Put the transformed cells into 42℃ water bath for 60 seconds.
- Then place the cells in ice for 2 minutes.
- Add 1ml LB to the cells and mixed.
- Put the eppendorf tube in 37℃ water bath and incubate for an hour.
- Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.
- While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.
- Incubate at 37℃ incubater for 16~18 hours.
PCR
1. Dissolve the primers in water to have the concentration of 10nM.
2. PCR reaction mixer：
Template DNA（10ng/μl） 5 10× PCR buffer 2 10× dNTP（2mM） 2 forward primer（10μM） 0.5 reverse primer（10μM） 0.5 Pfu DNA polymerase（2Kb） 0.1 PCR water 9.9 _______________________________________ Total 20 μl
3. Put the reaction mixer in a PCR tube.
4.The PCR program is as follow :
- 4.1
- 94℃ 30 seconds
- 60℃ 30seconds
- 72℃ 2 minutes
- Cycle 9 times
- 4.2
- 94℃ 30 seconds
- 55℃ 30seconds
- 72℃ 2 minutes
- Cycle 34 times
Extend PCR product at 72℃ for 10 minutes
5. The PCR product was examed by electrophoresis in 1％ agarose.
T-A cloning
Take an eppendorf tube and add following component one by one and mix well,incubated at 4℃ overnight.
2x ligase buffer 7.5μl Insert(Aeq.-GFP) 5.5μl Vector(pGEM-T-easy) 1μl T4 DNA exp 3/12 1μl _________________________________ total 15μl
Cloning PCR(To verify the presence of our gene of interest)
- Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.
- Discard the supernatant
- Add 500μl ddH20, Vortex
- Boil for 20 min.
- Take 5μl of above bacterial lysate, and add 10x dNTP 2μl, 10x Buffer 2μl, forward primer（10μM）0.5μl reverse primer（10μM）0.5μl, Taq DNA polymerase 0.1μl, ddH20
- Use the PCR to amplify our product：PCR program
95℃ 4 min 94℃ 30seconds 55℃ 40seconds 72℃ 2 min Cycle 34 times 25℃ 2 min
7. The PCR product was examed by electrophoresis in 1％ agarose.
Plasmid extraction(Homemade)
- Transfer 1.5ml of bacterial culture to each well(24 wells plate)
- pellet cells by centrifuging at 33000 rpm for 10min at 4℃
- carefully remove the supernatant
- add 100μl of Solution 1 (*)to each well, and vortex
- add 200μl of Solution 2 (*)to each well, and mix gently
- add 150μl of Solution 3 (*)to each well
- centrifuge 3000rpm at 4℃ for 10min
- add 1 ml 100% EtOH to new well
- Transfer the supernatant to the new well, containing 100% EtOH.
- centrifuge 3300rpm at 4℃ for 30min.
- carefully remove the supernatant.
- add 75% EtOH to wash pellets, then remove the supernantant, then air dry.
- add 40μl ddH2O to each well, to dissolve with plasmid DNA
- store the plate in 4℃
(*)Sol 1: 10m Miris/0.5mM HEDTA, PH 7.4 buffer.
Sol 2: 0.2N NaOH/1% SDS
Sol 3: 3M KOAC/HOAC
Digestion
Digestion mixture
DNA 20μl 10x buffer 3μl Enzyme 1μl RNase H2O 6μl __________________________ Total 30μl
3. The order for adding materials to wells is from plenty to less 4. Place the plate in 37℃water bath overnight
