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

Line 144: Line 144:
  
  
 +
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°
  
  

Revision as of 01:13, 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°

Biosensor Notebook

Kaempferol Notebook

Plasmid Maintenance Notebook