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Team:British Columbia/Notebook/Flavours
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'''Experrimenter:''' Anna Müller | '''Experrimenter:''' Anna Müller | ||
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'''Aim:''' PCR COMT from cleaned up DNA for digest | '''Aim:''' PCR COMT from cleaned up DNA for digest | ||
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'''Results:'''Bands were seen on a gel. | '''Results:'''Bands were seen on a gel. | ||
Revision as of 02:55, 19 September 2013
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Team Cinnamaldehyde & Vanillin
Research Designs and Methods
The goal for cloning is to place each gene in the cinnamaldehyde pathways under a constitutive pTET and arabinose promoter pBAD for future characterization of each part. Our final construct should consist of a single pSB1C3 plasmid with all the genes inserted along with its ribosomal binding site and terminator:
Primers for the PAL/TAL, Ligase, and reductase genes are designed to yield PCR products containing all biobrick cut sites in the correct order: Eco R1, Xba 1, Spe1, and PstI. These PCR products could then be digested for ligation into available plasmids containing promoters (with or without rbs) and terminators.
June 28th
Transformation of 4-coumarate-CoA Ligase (4CL) and caffeic acid-O-methyl transferase (COMT)
Experimenter: Joe
Aim: Transform biobricks 4CL ( 2013 Distribution Kit, Plate 2, Well 17P) and COMT (2013 Distribution Kit, Plate 5, Well 19D) for miniprep and later amplification through PCR
Results: Transformation done according transformation procedures. Competent cells were plated on LB+ Amp plates and grown overnight at 37C. Only 4-CL grew overnight. No colonies were observed for COMT. Inoculated a colony of 4CL for miniprep
Rhodobacter sphaeroides Inoculation
Experimenter: Joe
Aim: A colony of Rhodobacter sphaeroides was inoculated in 5mL LB culture for DNA extraction
July 30th
Miniprep 4CL
Experimenter: Joe
Aim: Miniprep 5mL innoculation of 4CL with Qiagen Miniprep Kit
July 4th
PCR of 4-CL
Experimenter: Joe
Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL
Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.
July 4th
PCR of 4-CL
Experimenter: Joe
Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL
Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.
Retransformation COMT
Experimenter: Joe
Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL
Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.
July 4th
PCR of 4-CL
Experimenter: Joe
Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL
Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.
August 19th
Experimenter: Anna Müller
Aim: Check if Fisal’s ligations were successful: TAL in PSBIC3 and TAL with arabinose promoter with colony PCR
Results: 2 colonies were picked for each. No bands were seen on the gel. Ligations were unsuccessful.
August 21st
Experimenter: Anna Müller
Aim: Ligate PAL and COMP to a constitutive promoter with RBS (.22A)
Results: After transforming, 2 colonies were picked and PCR’d (21st Aug. and 26th Aug.). The gel confirmed the presence
of both PAL (2100bp) and COMT (1001bp).
August 27th
Experimenter: Anna Müller
Aim: Digest PAL (cPCR product) with EcorI and Spe I so it can be ligated to the terminator.
Results: No bands were seen on the gel run. It was concluded to go back to the plate and inoculate from the colonies
that showed to contain PAL. The DNA then got cleaned up.
Experimenter: Anna Müller
Aim: Clean up COMT DNA to get it ready for the digest with Ecorl and Spe I
Results: Clean up successful.
Experrimenter: Anna Müller
Aim: PCR COMT from cleaned up DNA for digest
Results:Bands were seen on a gel.
