Team:TzuChiU Formosa/Protocol

  

Protocol
http://2009.igem.org/wiki/images/e/ea/Project.jpg

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)

 * 1) Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).
 * 2) Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.
 * 3) Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.
 * 4) Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)
 * 5) Spin down the bacteria at 4℃,3000 rpm for 10 min.
 * 6) Discard the supernatant and mix the cell pellet with 10ml FSB.
 * 7) Keep the cells on ice for 3~4 hours.
 * 8) Spin down, at 4℃,3000 rpm for 10 min.
 * 9) Discard the supernatant and mix the cell pellet with 5ml FSB.
 * 10) 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

 * 1) Take competent cell in an eppendorf tube from -80℃ freezer put in ice.
 * 2) Add 2ul plasmid to competent cell and place in ice for 5 minutes.
 * 3) Put the transformed cells into 42℃ water bath for 60 seconds.
 * 4) Then place the cells in ice for 2 minutes.
 * 5) Add 1ml LB to the cells and mixed.
 * 6) Put the eppendorf tube in 37℃ water bath and incubate for an hour.
 * 7) Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.
 * 8) 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.
 * 9) 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

Colony PCR(To verify the presence of our gene of interest)

 * 1) Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.
 * 2) Discard the supernatant
 * 3) Add 500μl ddH20, Vortex
 * 4) Boil for 20 min.
 * 5) 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
 * 6) 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)
(*)Sol 1: 10m Miris/0.5mM HEDTA, PH 7.4 buffer. Sol 2: 0.2N NaOH/1% SDS Sol 3: 3M KOAC/HOAC
 * 1) Transfer 1.5ml of bacterial culture to each well(24 wells plate)
 * 2) pellet cells by centrifuging at 33000 rpm for 10min at 4℃
 * 3) carefully remove the supernatant
 * 4) add 100μl of Solution 1 (*)to each well, and vortex
 * 5) add 200μl of Solution 2 (*)to each well, and mix gently
 * 6) add 150μl of Solution 3 (*)to each well
 * 7) spin down 3000rpm at 4℃ for 10min
 * 8) add 1 ml 100% EtOH to new well
 * 9) Transfer the supernatant to the new well, containing 100% EtOH.
 * 10) spin down 3300rpm at 4℃ for 30min.
 * 11) carefully remove the supernatant.
 * 12) add 75% EtOH to wash pellets, then remove the supernantant, then air dry.
 * 13) add 40μl ddH2O to each well, to dissolve with plasmid DNA
 * 14) store the plate in 4℃

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

Ligation
Take an eppendorf tube and add following component one by one and mix well,incubated at 16℃ overnight.

Insert(Aeq.-GFP)	7.5μl Vector(pSB1A3) 	5.5μl 10X ligase buffer	1μl T4 DNA ligase  	1μl _________________________________ total			30μl