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Team:Hong Kong HKUST/wetlab/notebook
From 2014.igem.org
Notebook
June 2013
July 2013
Week 2
July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 PfadBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (PfadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 PfadBA restriction check, gel extraction and purification
July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 PfadBA 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 PfadBA 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 PfadBA and BBa_J176171
· BBa_J52034 (fadR) inoculation
· Digestion for BBa_K817002 PfadBA extraction
Gel check for BBa_K817002 PfadBA extraction
July 11
Wet lab
· Gel purification for BBa_K817002 PfadBA 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 PfadBA
Dry Lab
Primers design for pCMV cloning for fadR expression
July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 PfadBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (PfadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 PfadBA restriction check, gel extraction and purification
July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 PfadBA 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 PfadBA 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 PfadBA and BBa_J176171
· BBa_J52034 (fadR) inoculation
· Digestion for BBa_K817002 PfadBA extraction
Gel check for BBa_K817002 PfadBA extraction
July 11
Wet lab
· Gel purification for BBa_K817002 PfadBA 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 PfadBA
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
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
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
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
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
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
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