Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and
research findings. The notebook for Pneuumonsensor and Riboregulator are displayed separately.
Pneumosensor
June 2013
Week 1
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 2
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 3
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 4
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 5
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
July 2013
Week 1
July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of P fadBA due chloramphenicol plates contamination
July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPAR-alpha promoter amplification
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
July 4
Wet lab
· Inoculation of BBa_K817002 P fadBA
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Ran gel for digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78
July 5
Wet lab
· Extraction of BBa_K817002 (P fadBA)
· Inoculation of pEGFP-N1
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter
· Poured new LB-Kanamycin plates
· Transformation of BBa_J176171
Dry lab
· Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector
· Primer redesign for PPAR-alpha promoter and FABP1 promoter
Week 2
July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 P fadBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (P fadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 P fadBA restriction check, gel extraction and purification
July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 P fadBA and pEGFP-N1 by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of BBa_J176171 for P fadBA Ase1 and BamH1; FABP1 promoter by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· BBa_J52034 ( fadR) transformation
July 10
Wet lab
· Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter
· PCR for FABP1 promoter, PCR replication
· BBa_J176171 vector dephosphorylation by antartic phosphatase
· Ligation of BBa_K817002 and P fadBA and BBa_J176171
· BBa_J52034 ( fadR) inoculation
· Digestion for BBa_K817002 P fadBA extraction
Gel check for BBa_K817002 P fadBA extraction
July 11
Wet lab
· Gel purification for BBa_K817002 P fadBA extraction
· Gel check for FABP1 promoter PCR product
· PCR clean-up
· BBa_J52034 ( fadR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPAR-alpha promoter
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions
July 12
Wet lab
· Digestion of FABP1 promoter PCR product
· Ran gel for PCR check of PPAR-alpha promoter
· Digest pEGFP-n1 for BBa_J52034 fadR
· Ran gel for pEGFP-n1 for BBa_J52034 fadR followed by gel purification
· BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and P fadBA
Dry Lab
Primers design for pCMV cloning for fadR expression
Week 3
July 15
Wet lab
· Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 promoter, EGFP, and BBa_J176171 ligation
· FABP1 promoter+EGFP +BBa_J176171 ligation restriction check
July 16
Wet Lab
· Miniprep for full construct of FABP1 promoter and pEGFP-N1
· fadR and pEGFP-N1 Backbone parts ligation
· Plasmids extraction for pCMV cloning for fadR
· BBa_K817002 PfadBA promoter extraction by EcoR1 and Pst1 HF
· BBa_J52034 restriction by EcoR1 and Pst1 HF
· Inoculations for full construct of FABP1 promoter and pEGFP-N1
· Streak colonies containing the right construct for FABP1 promoter
July 17
Wet lab
· Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep
· Repeat fadR and PfadBA constructs
· Digest pEGFP-n1 and BBa-J176171 for fadR
· Digestion for BBa_K817002 (PfadBA) extraction
· Gel check and gel extraction for BBa_J176171 for fadR and BBa_K817002 (PfadBA)
July 18
Wet lab
· Gel purification for all digested products
· FABP1 promoter digestion check for whole construct prior transfection
· PfadBA vector de phosphorylation and ligation with EGFP and BBa_J176171
· Transformation of PfadBA+EGFP+BBa_J176171
July 19
Wet lab
· FABP1 promoter preparation for transfection
· Ligation of PfadBA and BBa_J176171 followed by transformation
· PCR for PPAR-alpha promoter cloning
Dry lab
· Design for characterization of the transfected cells with FABP1 construct
· Review for Multiple Sites Mutagenesis
· Ordered primers for pCMV cloning from pEGFP-N1
Week 4
July 22
Wet lab
· FABP1 construct given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of PfadBA and BBa_J176171
· Ran gel for PPAR-alpha promoter PCR products
July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1 promoter
· PfadBA gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation
· Digestion of pEGFP-N1 for fadR, followed by gel check and extraction
July 24
Wet lab
· DNA purification from gel extraction for digested pEGFP-N1
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPAR-alpha promoter with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of PfadBA ligation colonies
July 25
Wet lab
· Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase
· Minprep of PfadBA+EGFP+BBa_J176171 ligation colonies, followed by digestion check
July 26
Wet lab
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel before parental string digestion of mutagenesis products
· Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check
· Plasmids preparation according to transfection requirements, construct and controls
Week 5
July 30
Wet lab
· PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1
· BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b
· PCR for PPAR-alpha promoter with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1
July 31
· Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up
· PCR for PPAR-alpha promoter with reference primers using Vent polymerase
· Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase
August 2013
Week 1
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 2
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 3
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 4
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 5
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
September 2013
Week 1
August 2
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 2
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 3
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 4
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 5
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Riboregulator
June 2013
Week 1
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 2
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 3
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 4
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 5
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
June xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
July 2013
Week 1
July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of P fadBA due chloramphenicol plates contamination
July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPAR-alpha promoter amplification
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
July 4
Wet lab
· Inoculation of BBa_K817002 P fadBA
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Ran gel for digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78
July 5
Wet lab
· Extraction of BBa_K817002 (P fadBA)
· Inoculation of pEGFP-N1
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter
· Poured new LB-Kanamycin plates
· Transformation of BBa_J176171
Dry lab
· Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector
· Primer redesign for PPAR-alpha promoter and FABP1 promoter
Week 2
July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 P fadBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (P fadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 P fadBA restriction check, gel extraction and purification
July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 P fadBA and pEGFP-N1 by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of BBa_J176171 for P fadBA Ase1 and BamH1; FABP1 promoter by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· BBa_J52034 ( fadR) transformation
July 10
Wet lab
· Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter
· PCR for FABP1 promoter, PCR replication
· BBa_J176171 vector dephosphorylation by antartic phosphatase
· Ligation of BBa_K817002 and P fadBA and BBa_J176171
· BBa_J52034 ( fadR) inoculation
· Digestion for BBa_K817002 P fadBA extraction
Gel check for BBa_K817002 P fadBA extraction
July 11
Wet lab
· Gel purification for BBa_K817002 P fadBA extraction
· Gel check for FABP1 promoter PCR product
· PCR clean-up
· BBa_J52034 ( fadR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPAR-alpha promoter
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions
July 12
Wet lab
· Digestion of FABP1 promoter PCR product
· Ran gel for PCR check of PPAR-alpha promoter
· Digest pEGFP-n1 for BBa_J52034 fadR
· Ran gel for pEGFP-n1 for BBa_J52034 fadR followed by gel purification
· BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and P fadBA
Dry Lab
Primers design for pCMV cloning for fadR expression
Week 3
July 15
Wet lab
· Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 promoter, EGFP, and BBa_J176171 ligation
· FABP1 promoter+EGFP +BBa_J176171 ligation restriction check
July 16
Wet Lab
· Miniprep for full construct of FABP1 promoter and pEGFP-N1
· fadR and pEGFP-N1 Backbone parts ligation
· Plasmids extraction for pCMV cloning for fadR
· BBa_K817002 PfadBA promoter extraction by EcoR1 and Pst1 HF
· BBa_J52034 restriction by EcoR1 and Pst1 HF
· Inoculations for full construct of FABP1 promoter and pEGFP-N1
· Streak colonies containing the right construct for FABP1 promoter
July 17
Wet lab
· Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep
· Repeat fadR and PfadBA constructs
· Digest pEGFP-n1 and BBa-J176171 for fadR
· Digestion for BBa_K817002 (PfadBA) extraction
· Gel check and gel extraction for BBa_J176171 for fadR and BBa_K817002 (PfadBA)
July 18
Wet lab
· Gel purification for all digested products
· FABP1 promoter digestion check for whole construct prior transfection
· PfadBA vector de phosphorylation and ligation with EGFP and BBa_J176171
· Transformation of PfadBA+EGFP+BBa_J176171
July 19
Wet lab
· FABP1 promoter preparation for transfection
· Ligation of PfadBA and BBa_J176171 followed by transformation
· PCR for PPAR-alpha promoter cloning
Dry lab
· Design for characterization of the transfected cells with FABP1 construct
· Review for Multiple Sites Mutagenesis
· Ordered primers for pCMV cloning from pEGFP-N1
Week 4
July 22
Wet lab
· FABP1 construct given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of PfadBA and BBa_J176171
· Ran gel for PPAR-alpha promoter PCR products
July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1 promoter
· PfadBA gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation
· Digestion of pEGFP-N1 for fadR, followed by gel check and extraction
July 24
Wet lab
· DNA purification from gel extraction for digested pEGFP-N1
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPAR-alpha promoter with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of PfadBA ligation colonies
July 25
Wet lab
· Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase
· Minprep of PfadBA+EGFP+BBa_J176171 ligation colonies, followed by digestion check
July 26
Wet lab
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel before parental string digestion of mutagenesis products
· Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check
· Plasmids preparation according to transfection requirements, construct and controls
Week 5
July 30
Wet lab
· PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1
· BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b
· PCR for PPAR-alpha promoter with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1
July 31
· Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up
· PCR for PPAR-alpha promoter with reference primers using Vent polymerase
· Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase
August 2013
Week 1
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 2
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 3
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 4
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 5
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Aug xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
September 2013
Week 1
August 2
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 2
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 3
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 4
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Week 5
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
Sept xx
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
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