Team:NYMU-Taipei/Notebook
HAIR TO STAY
JUNE
week1
- Plasmid pET32a was extracted from BL21 DE3.
- We prepared competent cell solution buffer and made competent cell of BL21 and DH5-alpha.
- Plasmid pET32a、pET29b were transfected into competent cell DH5-alpha.
- Plasmid pET32a was extracted from competent cell DH5-alpha
- Plasmid pET32a was checked with restriction digestion(NcoI and NotI).
- Plasmid CMV-Mycj-YFP, pCMV-Mycj-CFP and pET29b were transfected into competent cell DH5-alpha
JULY
week2
FRET SYSTEM
- Plasmid pSB1C3（ECFP），pSB1C3(SYFP2) and pSB1C3(YFP) were transformed into competent cell DH5-alpha and BL21.
CELL SYSTEM
- 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 SYSTEM
- Plasmid pSB1C3-ECFP/EYFP/SYFP2/CYFP were extracted from competent cell BL21 and checked with restriction digestion(NcoI and EcoRI).
- Plasmid pCMV-Mycj3-ECFP was checked with restriction digestion(XbaI and XhoI).
- The Plasmid with CyPet and YPet was transformed into competent cell DH5-alpha.
- ECFP/EYFP/SYFP2 were amplified by PCR and extracted from agarose gel.
CELL SYSTEM
- 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 SYSTEM
- ECPF/EYFP/SYFP2 were amplified by PCR.
- YPet/CyPet pre/post were amplified by PCR.
- Plasmid GEX2T-SUMO1/cDNA3.1-T7-Ubc9/TriEx4-Rac1-2G were transfected into competent cell DH5-alpha.
- Plasmid GEX2T-SUMO1/cDNA3.1-T7-Ubc9/TriEx4-Rac1-2G were extracted from competent cell DH5-alpha.
week5
FRET SYSTEM
- CyPet/YPet/ ECFP/ EYFP/SYFP2 inserted intoplasmid pET32a.
- The plasmids pET32a+ECFP/EYFP/SYFP2/CyPet/YPet were transformed into competent cell BL21.
- SUMO1 and Ubc9 from pGEX2T and pcDNA3.1-T7 were amplified by PCR.
- Plasmids pUC57-H7,G5(VHH),pET32a-ECFP/EYFP/SYFP2/CyPet/YPet were extracted from DH5-alpha.
- Plasmid Fluorescence Protein+pET32a was checked with restriction digestion(BamHI and SalI).
CELL SYSTEM
- 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 SYSTEM
- CyPet, YPet/ ECFP/ EYFP/SYFP2 post and plasmid pET32a inserted into plasmid pET32a.
- Plasmids pET32a+FP(ECFP/EYFP/SYFP2/CyPet/YPet) were transformed into BL21.
- Plasmid pUC57-insert was checked with restriction digestion(XbaI and XhoI).
- The plasmid with LRP6 was transformed into competent cell DH5-alpha and BL21.
- Plasmids pET32a and pUC57-FPFSkel1 were digested with restriction enzymes(XbaI and XhoI）
- Plasmid pET32a was ligated with FPFSkel1.
- Plasmid pET32a-FPFSkel1 was transformed into competent cell BL21.
- His-tag was annealed.
- The plasmid with LRP6 was extracted from competent cell BL21.
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
- 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 SYSTEM
CELL SYSTEM
OCTOBER
week14
FRET SYSTEM
CELL SYSTEM
week15
FRET SYSTEM
CELL SYSTEM
week16
FRET SYSTEM
CELL SYSTEM