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

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)