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)