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Revision as of 13:54, 21 October 2009 (view source) LXZ (Talk | contribs) ← Older edit		Latest revision as of 01:01, 22 October 2009 (view source) LXZ (Talk | contribs)
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-	====T-A cloning protocol====	+	====T-A cloning====
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Line 112:		Line 115:
-	====Cloning PCR(To verify the presence of our gene of interest)====	+	====Colony PCR(To verify the presence of our gene of interest)====
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		+
		+
		+
		+
	#Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.		#Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.
	#Discard the supernatant		#Discard the supernatant
Line 126:		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.	+	7. The PCR product was examed by electrophoresis in 1％ agarose.
		+
		+	====Plasmid extraction(Homemade)====
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		+
		+
		+
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		+	#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
		+	#spin down 3000rpm at 4℃  for 10min
		+	#add 1 ml 100% EtOH to new well
		+	#Transfer the supernatant to the new well, containing 100% EtOH.
		+	#spin down 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
		+
		+
		+
		+	====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

---

Latest revision as of 01:01, 22 October 2009

Contents 1 Protocol 1.1 Competent Cell (CP919-Cph8) 1.2 Tansformation Protocol 1.3 PCR 1.4 T-A cloning 1.5 Colony PCR(To verify the presence of our gene of interest) 1.6 Plasmid extraction(Homemade) 1.7 Digestion 1.8 Ligation

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)

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)

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℃

 (*)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

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

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