Difference between revisions of "Team:British Columbia/Notebook"

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pSB1C3 (Cm)
 
pSB1C3 (Cm)
 
          
 
          
−
 
+
<br>
 
    
 
    
  
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pSB1C3 (Cm)
 
pSB1C3 (Cm)
  
−
 
+
<br>
  
  
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pSB1C3 (Cm)
 
pSB1C3 (Cm)
  
−
 
+
<br>
  
  
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pSB1C3 (Cm)
 
pSB1C3 (Cm)
  
 +
<br>
  
  
 +
First, TAL and 4CL with RBS (ribosomal binding site) to combine them:
 +
<br>
 +
<ul>
 +
<li>4CL digest with EcoRI + SpeI </li>
 +
<li>TAL digest with EcoRI + Xbal</li>
 +
<li>Ligation of both digestion with T4 ligase </li>
 +
<li> E. coli transformation via heat shock method</li>
 +
<li>Spread on CMB-LB agar plate and incubate at 37 C° overnight </li>
 +
<li>PCR and gel electrophoresis to analyze if successful </li>
 +
<li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C </li>
 +
<li> Plasmid obtained</li>
 +
</ul>
  
−
First, TAL and 4CL with RBS (ribosomal binding site) to combine them:
 
−
- 4CL digest with EcoRI + SpeI
 
−
- TAL digest with EcoRI + Xbal
 
−
- Ligation of both digestion with T4 ligase
 
−
- E. coli transformation via heat shock method
 
−
- Spread on CMB-LB agar plate and incubate at 37 C° overnight
 
−
- PCR and gel electrophoresis to analyze if successful
 
−
- Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
 
−
- Plasmid obtained
 
 
Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):  
 
Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):  
−
- CHS digest with EcoRI and SpeI  
+
<br>
−
- CHI digest with EcoRI and Xbal  
+
<ul>
−
- Ligation of both digestion with T4 ligase  
+
<li>CHS digest with EcoRI and SpeI </li>
−
- E. coli transformation via heat shock method  
+
<li> CHI digest with EcoRI and Xbal </li>
−
- Spread on CMB-LB agar plate at 37c° overnight  
+
<li> Ligation of both digestion with T4 ligase </li>
−
- PCR and gel electrophoresis to analyze if successful  
+
<li> E. coli transformation via heat shock method </li>
−
- Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
+
<li> Spread on CMB-LB agar plate at 37c° overnight </li>
−
- Plasmid obtained
+
<li> PCR and gel electrophoresis to analyze if successful </li>
 +
<li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°</li>
 +
<li> Plasmid obtained</li>
 +
</ul>
 +
 
 
Third, combine the two plasmids previously prepared:
 
Third, combine the two plasmids previously prepared:
−
- 4CL – TAL digest by EcoRI and SpeI
+
<br>
−
- CHS-CHI with EcoRI and Xbal  
+
<ul>
−
- Dephosphorylate CHS-CHI  
+
<li> 4CL – TAL digest by EcoRI and SpeI</li>
−
- Ligate the two together with T4 ligation  
+
<li>CHS-CHI with EcoRI and Xbal </li>
−
- Add GFP via digestion and then ligation*  
+
<li> Dephosphorylate CHS-CHI </li>
−
- E. coli transformation via heat shock method  
+
<li> Ligate the two together with T4 ligation </li>
−
- PCR and gel electrophoresis to analyze if successful  
+
<li> Add GFP via digestion and then ligation* </li>
−
- Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°
+
<li> E. coli transformation via heat shock method </li>
−
 
+
<li> PCR and gel electrophoresis to analyze if successful </li>
 +
<li> Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°</li>
 +
</ul>
  
  

Revision as of 04:48, 17 October 2018

Naringenin Notebook

We kindly received the following from UBC iGEM distribution kit of 2018:
Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
4CL BBa_K801093 ~1700 BP Spring 2018 Distribution Plate 2, Well 17D pSB1C3 (Cm)
RBS+TAL BBa_K1033000 ~1600 Spring 2018 Distribution Plate 4, Well 6C pSB1C3 (Cm)
First, TAL and 4CL with RBS (ribosomal binding site) to combine them:

  • 4CL digest with EcoRI + SpeI
  • TAL digest with EcoRI + Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate and incubate at 37 C° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
  • Plasmid obtained
Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):
  • CHS digest with EcoRI and SpeI
  • CHI digest with EcoRI and Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate at 37c° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
  • Plasmid obtained
Third, combine the two plasmids previously prepared:
  • 4CL – TAL digest by EcoRI and SpeI
  • CHS-CHI with EcoRI and Xbal
  • Dephosphorylate CHS-CHI
  • Ligate the two together with T4 ligation
  • Add GFP via digestion and then ligation*
  • E. coli transformation via heat shock method
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°
Protocol for cloning promoter-RBS-4CL in pSB1C3 A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)

Biosensor Notebook

Kaempferol Notebook

Plasmid Maintenance Notebook