Team:Tokyo Metropolitan/Project/Pattern/Protocol

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Contents 1 PCR with Pho DNA Polymerase (NIPPON GENE) 1.1 ＜Materials＞ 1.2 <Process> 2 PCR with Tag DNA Polymerase (NIPPON GENE) 2.1 ＜Materials＞ 2.2 <Process> 3 DNA Ligation with Ligation-convenience kit (NIPPON GENE) 3.1 <Materials> 3.2 <Process> 4 DNA extraction 4.1 ＜Materials＞ 4.2 ＜Process＞ 5 DNA extraction with ISOPLANT(NIPPON GENE) 5.1 ＜Materials＞ 5.2 ＜Process＞ 6 Plasmid extraction from E-coli 6.1 ＜Materials＞ 6.2 ＜Process＞ 7 Transformation with ECOS competent E-coli(NIPPON GENE) 7.1 ＜Materials＞ 7.2 ＜Process＞ 8 Electrophoresis 8.1 ＜Materials＞ 8.2 ＜Process＞ 8.3 ＜Marker＞ 9 DNA Digestion 9.1 <Material> 9.2 ＜Process＞ 10 Ligation with Ligation-convenience kit (NIPPON GENE) 10.1 <Materials> 10.2 <Process> 11 DNA extraction with ISOPLANT(NIPPON GENE) 11.1 ＜Materials＞ 11.2 ＜Process＞ 12 About Sample

PCR with Pho DNA Polymerase (NIPPON GENE)

＜Materials＞

• 10x reaction buffer
  
• Forward primer (starting with 20µM)
  
• Reverse primer (starting with 20µM)
  
• Pho DNA Polymerase (starting with 2.5U/µl)
  
• dNTP (starting with 2.5mM)
  
• DW
  
• DNA samples
  

<Process>

1. Make the pellet with pre-colony
   
2. Thaw all required reagents completely and put them on ice. Mix all reagents well by inversion and spin them down prior to pipeting.
   
3. Prepare the reaction mix to correct for dispensing losses prepare an excess of reaction mix (for example, a 100 reactions mix for 96 reactions)
   
   • 10x reaction buffer 12.5μl
     
   • Forward primer 2.5μl (starting with 20µM)
     
   • Reverse primer 2.5μl (starting with 20µM)
     
   • Pho DNA Polymerase 2.5µl (starting with 2.5U/µl)
     
   • dNTP 20μl (starting with 2.5mM)
     
   • DW 85µl
     
   • Total reaction Mix 125μl
     
4. Add all components together, except for the template. Mix thoroughly by inversion. Spin down.
   
5. Add the template DNA(pellet or Plasmid) for your samples in to PCR tubes
   
6. Add 50μl of the reaction mix per tube and mix gently on a stirrer or spin down. Ensure that no bubbles are present in the reaction tube. Reaction set up can be done at room temperature.
   
7. Program your Real-Time Thermocycler w/ “fast block” using the following recommended FAST parameters:
   
   • 95°C 5min.
     
   • 95°C 30sec. 30cycle
     
   • 55°C 30sec. 30cycle
     
   • Tm-5°C 2~4min. 30cycle
     
   • 72°C 5min.
     
   • 4°C ∞　
     

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PCR with Tag DNA Polymerase (NIPPON GENE)

＜Materials＞

• 10x reaction buffer
  
• Forward primer (starting with 20µM)
  
• Reverse primer (starting with 20µM)
  
• Tag DNA Polymerase (starting with 2.5U/µl)
  
• dNTP (starting with 2.5mM)
  
• DW
  
• DNA samples
  

<Process>

1. Make the pellet with pre-culture
   
2. Thaw all required reagents completely and put them on ice. Mix all reagents well by inversion and spin them down prior to pipeting.
   
3. Prepare the reaction mix to correct for dispensing losses prepare an excess of reaction mix (for example, a 100 reactions mix for 96 reactions)
   
   • 10x reaction buffer 12.5μl
     
   • Forward primer 2.5μl (starting with 20µM)
     
   • Reverse primer 2.5μl (starting with 20µM)
     
   • Tag DNA Polymerase 1.0µl (starting with 2.5U/µl)
     
   • dNTP 10μl (starting with 2.5mM)
     
   • DW 96.5µl
     
   • Total reaction Mix 125μl
     
4. Add all components together, except for the template. Mix thoroughly by inversion. Spin down.
   
5. Add the template DNA(pellet or Plasmid) for your samples in to PCR tubes
   
6. Add 50μl of the reaction mix per tube and mix gently on a stirrer or spin down. Ensure that no bubbles are present in the reaction tube. Reaction set up can be done at room temperature.
   
7. Program your Real-Time Thermocycler w/ “fast block” using the following recommended FAST parameters:
   
   • 95°C 5min.
     
   • 95°C 30sec. 30cycle
     
   • 55°C 30sec. 30cycle
     
   • Tm-5°C 　　2~4min. 30cycle
     
   • 72°C 5min.
     
   • 4°C ∞
     

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DNA Ligation with Ligation-convenience kit (NIPPON GENE)

<Materials>

• DNA solution
  
• 2 × Ligation Mix (Ligation-convenience kit from NIPPON GENE)
  

<Process>

1. Prepare 10 μl of DNA solution to contain DNA fragment with appropriate mole ratio against vector DNA.
   
2. Add 10μl of 2 × Ligation Mix to the DNA solution and mix well.
   
3. Ligation reaction. Incubate 5-30min at 16°C.
   
4. Apply DNA reaction mixture directly to transformation or in vitro packaging as it is.
   

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DNA extraction

＜Materials＞

• Binding Buffer (5M Guanidine Thiocyanate; 100mM Tris-HCl (pH7.0))
  
• Wash Buffer (10mM Tris-HCl (pH7.5))
  
• Silica gel solution
  
• TE (10mM Tris-HCl pH 8.0; 0.1mM EDTA)
  
• Sample
  

＜Process＞

1. Add 150µl of Binding Buffer to the tube and Vortex.
   
2. Add 10µl of Silica gel solution to the tube and Vortex.
   
3. Vortex every 1min for 5min.
   
4. Centrifuge on 12,000 rpm for 1min, and then aspirate the supernatant.
   
5. Add 200µl of Wash Buffer to tube and Vortex.
   
6. Centrifuge on 12,000 rpm for 30sec, and then aspirate the supernatant.
   
7. Add 200µl of Wash Buffer to tube and Vortex.
   
8. Centrifuge on 12,000 rpm for 30sec, and then aspirate the supernatant.
   
9. Dry the pellets in the speed-vac for 5min, or air dry.
   
10. Add 20µl of TE Buffer to the dried pellets
    

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DNA extraction with ISOPLANT(NIPPON GENE)

＜Materials＞

• Extraction Buffer
  
• Lysis Bufferl
  
• Sodium Acetate(pH5.2)
  
• 10mM Tris-HCl(pH8.0), 1mM EDTA
  
• 1mg/ml RNaseA
  
• E-coli culture
  
• TE(10mM Tris-HCl pH 8.0; 0.1mM EDTA)
  
• DW
  

＜Process＞

1. Centrifuge 1.5ml of E-coli culture at 4οC for 5min, and then aspirate the supernatant.
   
2. Add 0.3ml of Extraction Buffer to sample, and then vortex 1~2sec.
   
3. Add 0.15mlof Lysis Buffer to solution, and then vortex 5~6sec.
   
4. Incubate 15min at 50°C.
   
5. Add 0.15ml of Sodium Acetate(pH5.2) to solution, and then vortex 1~2sec.
   
6. Leave on ice for 15min.
   
7. Centrifuge solution at 4οC for 15min, and then aspirate the supernatant.
   
8. Add 0.3ml of H2O to the tube having the pellets, and then vortex thoroughly.
   
9. Add 0.6ml of EtOH to solution, and then vortex thoroughly.
   
10. Centrifuge solution for 10min, and then aspirate the supernatant.
    
11. Add 70%EtOH to the tube having the pellets.
    
12. Dry the pellets in the speed-vac for 5min, or air dry.
    
13. Add TE Buffer to the dried pellets
    

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Plasmid extraction from E-coli

＜Materials＞

• Binding Buffer (5M Guanidine Thiocyanate; 100mM Tris-HCl (pH7.0))
  
• Wash Buffer (10mM Tris-HCl (pH7.5))
  
• Silica gel solution
  
• TE (10mM Tris-HCl pH 8.0; 0.1mM EDTA)
  
• Sample
  

＜Process＞

1. Centrifuge 1ml of the E-coli pre-culture on 12,000 rpm for 30sec, and then aspirate the supernatant.
   
2. Add 100µl of the solution (50ｍM glucose; 10mM EDTA; 25ml Tris-HCl(pH 8.0)), and mix it gently
   
3. Add 200µl of the 0.2N NaOH; 1％ SDS, and mix it, and then incubate on ice for 5min.
   
4. Add 150µl of 3M Potassium Acetate (pH 4.8)(5M Acetic acid; 3M Potassium), and mix it, and then incubate on ice for 5min.
   
5. Centrifuge on 12,000 rpm for 5min.
   
6. Add 400µl of Binding Buffer and 10µl of Silica gel solution to another tube and Vortex.
   
7. Add the supernatant(in centrifuged tube) to solution of Binding Buffer and Silica gel, and Vortex.
   
8. Centrifuge on 12,000 rpm for 5min, and then aspirate the supernatant.
   
9. Add 800µl of Wash Buffer to tube and Vortex.
   
10. Centrifuge on 12,000 rpm for 30sec, and then aspirate the supernatant.
    
11. Add 800µl of Wash Buffer to tube and Vortex.
    
12. Centrifuge on 12,000 rpm for 30sec, and then aspirate the supernatant.
    
13. Dry the pellets in the speed-vac for 5min, or air dry.
    
14. Add 50µl of TE Buffer to the dried pellets
    

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Transformation with ECOS competent E-coli(NIPPON GENE)

＜Materials＞

• competent cell
  
• Plasmid solution
  
• LB plate(Amp)
  

＜Process＞

1. Keep a competent cell(JM109) at RT and then on ice until melting.
   
2. Add 5µl of the solution of ligated-recombinant plasmid DNA to the tube, which contains the competent cell, and mix it gently.
   
3. Keep the tube on ice for 5min.
   
4. Transfer the tube into 42°C water bath and keep it for 45sec.
   
5. Vortex a suspension and spread it on a LB plate.
   
6. Incubate the plate at 37°C for 12~16h.
   

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Electrophoresis

＜Materials＞

• TAE Buffer 100ml
  
• Agarose 1.0g
  
• Marker 5.0µl
  
• Dye 1.0µl
  
• DNA solution
  

＜Process＞

1. TAE Buffrer into agarose and Microwave for about 1min to dissolve the agarose.
   
2. Pour the agarose slowly into the gel board.
   
3. Set gel in a gel tank.
   
4. Pour the TAE Buffer into the gel tank.
   
5. Mix the marker, DW and Dye.
   
6. Pour the mixture sample into well.
   
7. Close the gel tank, switch on the power-source and run the gel at 100V for 20min.
   
8. Switch off and unplug the gel tank and carry the gel to the machine to look at the progress of the gel.
   

＜Marker＞

One of the marker we used is "OneSTEP Ladder 50/500"[http://www.nippongene.com/index/english/e_index.htm/ (NIPPONE GENE)]

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DNA Digestion

<Material>

• DNA solution
  
• Digest enzyme
  
• ×10 Buffer
  
• DW
  

＜Process＞

1. Add 5µl of DW and 2µl of ×10 Buffer into the tube, and mix it gently.
   
2. Add 10µl of DNA solution and 1µl of Digest enzyme into the tube, and mix it gently.
   
3. Incubate at 37°C for 5h.
   

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Ligation with Ligation-convenience kit (NIPPON GENE)

<Materials>

• DNA solution
• 2×Ligation Mix (Ligation-convenience kit NIPPON GENE)

<Process>

1. Prepare 10 μl of DNA solution to contain DNA fragment with appropriate mole ratio against vector DNA.
2. Add 10μl of 2 × Ligation Mix to the DNA solution and mix well.
3. Ligation reaction. Incubate 5-30min at 16°C.
4. Apply DNA reaction mixture directly to transformation or in vitro packaging as it is.

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DNA extraction with ISOPLANT(NIPPON GENE)

＜Materials＞

• Extraction Buffer 0.3ml
• Lysis Buffer 0.15ml
• Sodium Acetate(pH5.2) 0.15ml
• 10mM Tris-HCl(pH8.0), 1mM EDTA 0.1ml
• 1mg/ml RNaseA 1µl
• E-coli culture
• H20
• TE Buffer

＜Process＞

1. Centrifuge 1.5ml of E-coli culture at 4οC for 5min, and then aspirate the supernatant.
2. Add 0.3ml of Extraction Buffer to sample, and then vortex 1~2sec.
3. Add 0.15mlof Lysis Buffer to solution, and then vortex 5~6sec.
4. Incubate 15min at 50°C.
5. Add 0.15ml of Sodium Acetate(pH5.2) to solution, and then vortex 1~2sec.
6. Leave on ice for 15min
7. Centrifuge solution at 4οC for 15min, and then aspirate the supernatant.
8. Add 0.3ml of H2O to the tube having the pellets, and then vortex thoroughly.
9. Add 0.6ml of EtOH to solution, and then vortex thoroughly.
10. Centrifuge solution for 10min, and then aspirate the supernatant.
11. Add 70%EtOH to the tube having the pellets.
12. Dry the pellets in the speed-vac for 5min, or air dry.
13. Add TE Buffer to the dried pellets

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About Sample

No.			Digest enzyme	bp
1	BBa_K208017	Promoter-signal	AvrⅡ	266
2		CyaA	NheⅠ	2583
3	BBa_l12521	mRFP1-terminator	NheⅠ	861
4	BBa_K208017	Promoter	AvrⅡ	145
5	BBa_K208017	RBS-signal	SpeⅠ	103
6	BBa_l732901	LacZ	SpeⅠ	3093
7	BBa_l12521	terminator	NheⅠ	167
8		CRP	AvrⅡ	657
9	BBa_K156010	SBFP2	XbaⅠ	721
10	BBa_J23110	Promoter-RBS	SpeⅠ	70