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Team:Hong Kong HKUST/notebook/mod2
From 2013.igem.org
(Difference between revisions)
| Line 534: | Line 534: | ||
<div> | <div> | ||
<a href='#' class='head'>Week 5</a> | <a href='#' class='head'>Week 5</a> | ||
| - | <div class='content' align="left"> | + | <div class='content' align="left"> |
| + | <br><b>July 30</b> <br> | ||
| + | Wet lab<br> | ||
| + | · PPARa PCR cloning with new primers using Vent polymerase<br> | ||
| + | · Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1<br> | ||
| + | · BBa_J176171 vector de phosphorylation, followed by ligation with eGFP using T4 Ligase, then transformation into E. Coli strain DH10b<br> | ||
| + | · PCR for PPARa with new primers using Vent polymerase<br> | ||
| + | Dry lab<br> | ||
| + | · Discussion and re assessment of constructs related to pEGFP-N1<br> | ||
| + | |||
| + | |||
| + | <br><b>July 31</b> <br> | ||
| + | · Ran gel check for PPARa PCR cloning with new primers using Vent polymerase<br> | ||
| + | · PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up<br> | ||
| + | · PCR for PPARa with reference primers using Vent polymerase<br> | ||
| + | · Ran gel check for PPARa PCR cloning with reference primers using Vent polymerase<br><br> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 15:14, 19 September 2013
June 2013
Week 4
June 24
Wet lab
· Inoculation of pBlueScript KS(+) for training
· Autoclave basic materials
· Preparing LB
· LB-Ampicillin plates poured Dry lab
Protocols review
June 25
Wet lab
· Plasmid extraction of pBlueScript KS(+)
· LB-Chloramphenicol plates poured
Dry lab
· Primers design for FABP1, PPARa, GRP78
· Informal meeting
June 26
Wet lab
· Extraction of gDNA from HepG2 Cells for FABP1 and PPARa
· Transformation of pEGFP-N1 and BBa_K817002 (pFadBA)
June 27
Wet lab
· Inoculation of pEGFP-N1 and BBa_K817002 (pFadBA) for plasmid extraction
June 28
Wet lab
· Extraction of pEGFP-N1 and BBa_K817002 (pFadBA) plasmids by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and bamH1 for PPARa; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of c(pFadBA) by EcoR1 and Pst1
· Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (pFadBA)
June 30
Dry lab
Experiment planning and protocols revision for next week work
June 24
Wet lab
· Inoculation of pBlueScript KS(+) for training
· Autoclave basic materials
· Preparing LB
· LB-Ampicillin plates poured Dry lab
Protocols review
June 25
Wet lab
· Plasmid extraction of pBlueScript KS(+)
· LB-Chloramphenicol plates poured
Dry lab
· Primers design for FABP1, PPARa, GRP78
· Informal meeting
June 26
Wet lab
· Extraction of gDNA from HepG2 Cells for FABP1 and PPARa
· Transformation of pEGFP-N1 and BBa_K817002 (pFadBA)
June 27
Wet lab
· Inoculation of pEGFP-N1 and BBa_K817002 (pFadBA) for plasmid extraction
June 28
Wet lab
· Extraction of pEGFP-N1 and BBa_K817002 (pFadBA) plasmids by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and bamH1 for PPARa; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of c(pFadBA) by EcoR1 and Pst1
· Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (pFadBA)
June 30
Dry lab
Experiment planning and protocols revision for next week work
July 2013
Week 1
July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and bamH1 for PPARa; , Xba1 and BamH1 for FABP1
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of pFadBA due chloramphenicol plates contamination
July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPARa promoter amplification
· Digestion of pEGFP-N1 by Ase1 and bamH1 for PPARa; , Xba1 and BamH1 for FABP1
July 4
Wet lab
· Inoculation of BBa_K817002 pFadBA
· PCR for PPARa and FABP1 promoter cloning from gDNA
· Ran gel for digestion of pEGFP-N1 by Ase1 and bamH1 for PPARa; Xba1 and BamH1 for FABP1; Ase1 and Xhol1 for GRP78
July 5
Wet lab
· Extraction of BBa_K817002 (pFadBA)
· Inoculation of pEGFP-N1
· PCR for PPARa and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPARa and FABP1
· 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 PPARa and FABP1
July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and bamH1 for PPARa; , Xba1 and BamH1 for FABP1
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of pFadBA due chloramphenicol plates contamination
July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPARa promoter amplification
· Digestion of pEGFP-N1 by Ase1 and bamH1 for PPARa; , Xba1 and BamH1 for FABP1
July 4
Wet lab
· Inoculation of BBa_K817002 pFadBA
· PCR for PPARa and FABP1 promoter cloning from gDNA
· Ran gel for digestion of pEGFP-N1 by Ase1 and bamH1 for PPARa; Xba1 and BamH1 for FABP1; Ase1 and Xhol1 for GRP78
July 5
Wet lab
· Extraction of BBa_K817002 (pFadBA)
· Inoculation of pEGFP-N1
· PCR for PPARa and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPARa and FABP1
· 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 PPARa and FABP1
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 PPARa and FABP1 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 PPARa; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of BBa_J176171 for pFadBA Ase1 and BamH1; FABP1 by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPARa and FABP1 promoters cloning from gDNA
· BBa_J52034 (FADR) transformation
July 10
Wet lab
· Ran gel for PCR check of PPARa and FAPB1
· PCR for FABP1, 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 PCR product
· PCR clean-up
· BBa_J52034 (FADR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPARa
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions
July 12
Wet lab
· Digestion of FABP1 PCR product
· Ran gel for PCR check of PPARa
· 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 and FADR
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 PPARa and FABP1 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 PPARa; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of BBa_J176171 for pFadBA Ase1 and BamH1; FABP1 by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPARa and FABP1 promoters cloning from gDNA
· BBa_J52034 (FADR) transformation
July 10
Wet lab
· Ran gel for PCR check of PPARa and FAPB1
· PCR for FABP1, 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 PCR product
· PCR clean-up
· BBa_J52034 (FADR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPARa
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions
July 12
Wet lab
· Digestion of FABP1 PCR product
· Ran gel for PCR check of PPARa
· 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 and FADR
Dry Lab
Primers design for pCMV cloning for FADR expression
Week 3
July 15
Wet lab
· Ligation of FABP1 eGFP and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 eGFP and BBa_J176171 ligation
· FABP1+eGFP +BBa_J176171 ligation restriction check
July 16
Wet Lab
· Miniprep for full construct of FABP1 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 and pEGFP-N1
· Streak colonies containing the right construct for FABP1
July 17
Wet lab
· Plasmid extraction for FABP1+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 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 preparation for transfection
· Ligation of pFadBA and BBa_J176171 followed by transformation
· PCR for PPARa 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 eGFP and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 eGFP and BBa_J176171 ligation
· FABP1+eGFP +BBa_J176171 ligation restriction check
July 16
Wet Lab
· Miniprep for full construct of FABP1 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 and pEGFP-N1
· Streak colonies containing the right construct for FABP1
July 17
Wet lab
· Plasmid extraction for FABP1+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 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 preparation for transfection
· Ligation of pFadBA and BBa_J176171 followed by transformation
· PCR for PPARa 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 given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of pFadBA and BBa_J176171
· Ran gel for PPARa PCR products
July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1
· 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
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPARa with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of pFadBA ligation colonies
July 25
Wet lab
· Ran gel check PPARa 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
· 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 given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of pFadBA and BBa_J176171
· Ran gel for PPARa PCR products
July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1
· 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
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPARa with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of pFadBA ligation colonies
July 25
Wet lab
· Ran gel check PPARa 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
· 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
· PPARa 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 PPARa with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1
July 31
· Ran gel check for PPARa 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 PPARa with reference primers using Vent polymerase
· Ran gel check for PPARa PCR cloning with reference primers using Vent polymerase
July 30
Wet lab
· PPARa 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 PPARa with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1
July 31
· Ran gel check for PPARa 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 PPARa with reference primers using Vent polymerase
· Ran gel check for PPARa PCR cloning with reference primers using Vent polymerase
