Team:NYMU-Taipei/Notebook
HAIR TO STAY
JUNE
week1
- Plasmid pET32a was extracted from competent cell BL21 DE3.
- We prepared competent cell solution buffer and made competent cell DH5-alpha.
JULY
week2
FRET MODEL
CELL MODEL
- Plasmid pUC19 was transformed into competent cell DH5-alpha.
- Plasmid pUC19 was extracted from competent cell DH5-alpha and checked by gel electrophoresis.
- Plasmid pUC19 was checked with restriction digestion(DpnI, EcoRI and BamHI).
week3
FRET MODEL
CELL MODEL
- Dkk1 promoter was amplified from HEK293 genomic DNA by KOD and DreamTaq polymerase PCR.
- DKK1 promoter PCR amplification checked by running an agarose gel(PCR failed).
- DH5-alpha pUC19 was stored in glycerol stock.
week4
FRET MODEL
week5
FRET MODEL
CELL MODEL
- KOD and DreamTaq polymerase PCR amplification of Dkk1 promoter from HEK293 genomic DNA.
- DKK1 promoter PCR amplification checked by running an agarose gel(Dream Taq success,KOD failed).
- troubleshooting of DKK1 promoter PCR amplification.
- KOD and DreamTaq polymerase PCR amplification of Dkk1 promoter from HEK293 genomic DNA.
- DKK1 promoter PCR amplification checked by running an agarose gel(Dream Taq success, KOD success).
AUGUST
week6
FRET MODEL
CELL MODEL
- DKK1 promoter junction PCR was conducted.
- DKK1 junction PCR product was extracted from agarose gel.
- DKK1 junction PCR product was amplified by KOD and DreamTaq polymerase PCR.
- Plasmid pUC19 was digested by restriction enzyme(EcoRI and BamHI).
- Dkk1 promoter was amplified from HEK293 genomic DNA by KOD and DreamTaq polymerase PCR.
- DKK1 junction PCR product was ligated with plasmid pUC19.
week7
FRET MODEL
CELL MODEL
- The ligation of Plasmid pUC19 and the DKK1 promoter was checked with restriction digestion(EcoRI and XbaI).
- The ligation of Plasmid pUC19 and the DKK1 promoter was checked with colony PCR.
week8
FRET MODEL
CELL MODEL
- Preparation for most of the materials including HEK293 (an immortalized kidney cancer cell), medium, and cultural dishes.
- Using HEK293 cells for basic training such as: refreshing mediums, subculturing cells, and freezing cells.
- Preparation for RT-PCR to get the 5-alpha reductase type 2 from HEK293 cells. We successfully extracted the cDNA of 5-alpha reductase type 2 for our following plasmid construction experiments.
week9
FRET MODEL
CELL MODEL
- Plasmid EGFP-LC3 was extracted from DH5-alpha.
- Plasmid EGFP was checked with restriction digestion(XhoI, NheI,MfeI and BamHI).
- CMV promoter was obtained from plasmid cDNA by using restriction enzyme(NheI and MfeI).
- CMV promoter was ligated with plasmid pUC19(EcoRI, XbaI, MfeI and NheI).
- improving cell culturing techniques and preparation for the arrival of immortalized human hair follicle dermal papilla cells.
SEPTEMBER
week10
FRET MODEL
CELL MODEL
- Reception of immortalized human hair follicle dermal papilla cells and testosterone from our generous collaborator - Dr. Chen Chih-Chiang.
- Test the properties of the DP cells before the upcoming transfection experiments.
- Test various transfection conditions by using HEK293 cells and PD cells.
week11
FRET MODEL
CELL MODEL
- Acquire suitable transfection conditions of HEK293 cells by testing with calcium phosphate transfection.
- The pEGFP-LC3 plasmid had already been tested through transfection
- DKK-1 promoter and secreting peptide attached reporter gene had also been cloned into pUC19.
week12
FRET MODEL
CELL MODEL
- Testing the difference between plasmid A(DKK-1 promoter+ mCherry+ BGA) and plasmid B( DKK-1 promoter+ ALB+ mCherry+ BGA) using calcium phosphate transfection technique.
- Adjusting the transfection conditions in DP cells.
week13
FRET MODEL
CELL MODEL
OCTOBER
week14
FRET MODEL
CELL MODEL
week15
FRET MODEL
CELL MODEL
week16
FRET MODEL
CELL MODEL