JUNE

week1

1. Plasmid pET32a was extracted from BL21 DE3.
2. We prepared competent cell solution buffer and made competent cell of BL21 and DH5-alpha.
3. Plasmid pET32a、pET29b were transfected into competent cell DH5-alpha.
4. Plasmid pET32a was extracted from competent cell DH5-alpha
5. Plasmid pET32a was checked with restriction digestion(NcoI and NotI).
6. Plasmid CMV-Mycj-YFP, pCMV-Mycj-CFP and pET29b were transfected into competent cell DH5-alpha

JULY

week2

FRET SYSTEM

1. Plasmid pSB1C3（ECFP），pSB1C3(SYFP2) and pSB1C3(YFP) were transformed into competent cell DH5-alpha and BL21.

CELL SYSTEM

1. Plasmid pUC19 was transformed into competent cell DH5-alpha.
2. Plasmid pUC19 was extracted from competent cell DH5-alpha and checked by gel electrophoresis.
3. Plasmid pUC19 was checked with restriction digestion(DpnI, EcoRI and BamHI).

week3

FRET SYSTEM

1. Plasmid pSB1C3-ECFP/EYFP/SYFP2/CYFP were extracted from competent cell BL21 and checked with restriction digestion(NcoI and EcoRI).
2. Plasmid pCMV-Mycj3-ECFP was checked with restriction digestion(XbaI and XhoI).
3. The Plasmid with CyPet and YPet was transformed into competent cell DH5-alpha.
4. ECFP/EYFP/SYFP2 were amplified by PCR and extracted from agarose gel.

CELL SYSTEM

1. Dkk1 promoter was amplified from HEK293 genomic DNA by KOD and DreamTaq polymerase PCR.
2. DKK1 promoter PCR amplification checked by running an agarose gel(PCR failed).
3. DH5-alpha pUC19 was stored in glycerol stock.

week4

FRET SYSTEM

1. ECPF/EYFP/SYFP2 were amplified by PCR.
2. YPet/CyPet pre/post were amplified by PCR.
3. Plasmid GEX2T-SUMO1/cDNA3.1-T7-Ubc9/TriEx4-Rac1-2G were transfected into competent cell DH5-alpha.
4. Plasmid GEX2T-SUMO1/cDNA3.1-T7-Ubc9/TriEx4-Rac1-2G were extracted from competent cell DH5-alpha.

week5

FRET SYSTEM

1. CyPet/YPet/ ECFP/ EYFP/SYFP2 inserted into plasmid pET32a.
2. The plasmids pET32a+ECFP/EYFP/SYFP2/CyPet/YPet were transformed into competent cell BL21.
3. SUMO1 and Ubc9 from pGEX2T and pcDNA3.1-T7 were amplified by PCR.
4. Plasmids pUC57-H7,G5(VHH),pET32a-ECFP/EYFP/SYFP2/CyPet/YPet were extracted from DH5-alpha.
5. Plasmid Fluorescence Protein+pET32a was checked with restriction digestion(BamHI and SalI).

CELL SYSTEM

1. KOD and DreamTaq polymerase PCR amplification of Dkk1 promoter from HEK293 genomic DNA.
2. DKK1 promoter PCR amplification checked by running an agarose gel(Dream Taq success,KOD failed).
3. troubleshooting of DKK1 promoter PCR amplification.
4. KOD and DreamTaq polymerase PCR amplification of Dkk1 promoter from HEK293 genomic DNA.
5. DKK1 promoter PCR amplification checked by running an agarose gel(Dream Taq success, KOD success).

AUGUST

week6

FRET SYSTEM

1. CyPet, YPet/ ECFP/ EYFP/SYFP2 post and plasmid pET32a inserted into plasmid pET32a.
2. Plasmids pET32a+FP(ECFP/EYFP/SYFP2/CyPet/YPet) were transformed into BL21.
3. Plasmid pUC57-insert was checked with restriction digestion(XbaI and XhoI).
4. The plasmid with LRP6 was transformed into competent cell DH5-alpha and BL21.
5. Plasmids pET32a and pUC57-FPFSkel1 were digested with restriction enzymes(XbaI and XhoI）
6. Plasmid pET32a was ligated with FPFSkel1.
7. Plasmid pET32a-FPFSkel1 was transformed into competent cell BL21.
8. His-tag was annealed.
9. The plasmid with LRP6 was extracted from competent cell BL21.

CELL SYSTEM

1. DKK1 promoter junction PCR was conducted.
2. DKK1 junction PCR product was extracted from agarose gel.
3. DKK1 junction PCR product was amplified by KOD and DreamTaq polymerase PCR.
4. Plasmid pUC19 was digested by restriction enzyme(EcoRI and BamHI).
5. Dkk1 promoter was amplified from HEK293 genomic DNA by KOD and DreamTaq polymerase PCR.
6. DKK1 junction PCR product was ligated with plasmid pUC19.

week7

FRET SYSTEM

1. The plasmid with LRP6,pET32a-New skel,pET32a-New skel,pET32a-New skel,pET32a-New skel,pET32a-New skel and pET32a-New skel were extracted from competent cell BL21 and DH5-alpha.
2. The plasmid with LRP6 was checked with restriction enzyme(EcoRI).
3. The plasmid pET32a-New skel,pET32a-New skel,pET32a-New skel,pET32a-New skel,pET32a-New skel and pET32a-New skel were checked with restriction enzymes(NheI and PstI).
4. The plasmid with Histag and pET32a-new skel were digested by restriction enzymes(KpnI and NheI).
5. The plasmid with Histag was ligated with plasmid pET32a-new skel.
6. The plasmid pET32a-New skel-Histag was transformed into competent cell BL21 and DH5-alpha.
7. The plasmids pUC57-VHH H7 and G5 were digested with restriction enzymes(KpnI and HindIII)
8. Plasmid pET32a-NewSkel-His tag was checked with restriction enzymes(HindIII and PstI).
9. The plasmid pET32a-New skel-Histag was extracted from competent cell DH5-alpha.
10. The plasmids with SUMO1, UBC9,Histag-new skel-pET32a and new skel-pET32a were digested with restriction enzymes(KpnI and BamHI)
11. The plasmids with SUMO1, UBC9,H7 and G5 were ligated with plasmid Histag-new skel-pET32a and plasmid new skel-pET32a and transformed into competent cell DH5-alpha.

CELL SYSTEM

1. The ligation of Plasmid pUC19 and the DKK1 promoter was checked with restriction digestion(EcoRI and XbaI).
2. The ligation of Plasmid pUC19 and the DKK1 promoter was checked with colony PCR.

week8

FRET SYSTEM

1. KOD polymerase PCR amplification of LRP6 E1 and E3, pre/post ECFP and pre/post SYFP2. ECFP、EYFP、Ypet、CyPet、SYFP protein expresses in E.coli was checked using a fluorescence microscope.
2. ECFPpost and SYFP2 post are ligated into plasmid pET32a.
3. KOD polymerase PCR amplification of LRP6 E1E2 and E3E4.
4. Plasmid pET32a-ECFP/SYFP2 is transformed into competent cell BL21.
5. Plasmid pET32a-New skel is transformed into competent cell DH5-alpha.

CELL SYSTEM

1. Preparation for most of the materials including HEK293 (an immortalized kidney cancer cell), medium, and cultural dishes.
2. Using HEK293 cells for basic training such as: refreshing mediums, subculturing cells, and freezing cells.
3. 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

1. The plasmid pUC57-VHH G5 and pUC57-VHH H7 were digested by restriction enzymes(KpnI and BamHI ).
2. The plasmid pUC57-VHH G5 and pUC57-VHH H7 were digested by restriction enzymes(KpnI and BamHI ).
3. CyPet post, YPet post and plasmid pET32a-NewSkel were digested with restriction enzymes(BamHI and SalI).
4. CyPet post, YPet post and His-tag post were ligated into New skel.
5. VHH(G5 &H7) were ligated into new skel.
6. Plasmid New skel-Histag/Cypet/Ypet were transformed into competent cell BL21

CELL SYSTEM

1. Plasmid EGFP-LC3 was extracted from DH5-alpha.
2. Plasmid EGFP was checked with restriction digestion(XhoI, NheI,MfeI and BamHI).
3. CMV promoter was obtained from plasmid cDNA by using restriction enzyme(NheI and MfeI).
4. CMV promoter was ligated with plasmid pUC19(EcoRI, XbaI, MfeI and NheI).
5. improving cell culturing techniques and preparation for the arrival of immortalized human hair follicle dermal papilla cells.

SEPTEMBER

week10

FRET SYSTEM

1. Plasmid pET32a-NewSkel-Histag was checked with restriction enzymes(XbaI and NheI).
2. Plasmid with New Skel and His tag were ligated.
3. KOD polymerase PCR amplification of CyPet post and YPet post.
4. New skel+CyPet was transformed into competent cell BL21

CELL SYSTEM

1. mCherry and BGA were cloned into pUC19 by using restriction enzyme SalI, BamHI and BamHI, HindIII respectively.
2. ALB (secreting peptide) was obtained by annealing PCR, and then fused with mCherry. The secreting mCherry was also cloned into pUC19 with SalI and BamHI.
3. mCherry and BGA were cloned into pUC19 simultaneously by digestion of pUC19 with SalI and HindIII and by two in one ligation.
4. Reception of immortalized human hair follicle dermal papilla cells and testosterone from our generous collaborator - Dr. Chen Chih-Chiang.
5. Test the properties of the DP cells before the upcoming transfection experiments.
6. Test various transfection conditions by using HEK293 cells and PD cells.

week11

FRET SYSTEM

1. tranform BL21 with Cypet+New skel, Ypet+ New skel, Ypet+ New skel, Histag+ New skel.
2. tranform DH5a with mCherry+BGA
3. tranform BL21 with Histag+post, Histag+post, post+CyPet/Ypet, post digested with KpnI NheI
4. CyPet post and YPet post were ligated together.
5. CyPet/YPet and post were ligated together.
6. Two-in-One Ligation of mCherry, BGA, pUC19
7. CyPet, YPet post were digest checked by restriction enzymes(PstI)
8. Histag post were digest checked by restriction enzymes(HindIII)
9. KOD polymerase PCR amplification of CyPet post and YPet post
10. KOD polymerase PCR amplification of EGFP mf+r
11. Junction PCR of EGFP SalIf and f

CELL SYSTEM

1. The secreting mCherry and BGA were also cloned into pUC19 simultaneously by digestion of pUC19 with SalI and HindIII and by two in one ligation.
2. CMV promoter region was first digested with MfeI and NheI from plasmid pcDNA3.1. However, the digestion quality and efficiency of enzyme NheI was found less capable than expected by the running the agarose gel electrophoresis. CMV promoter was then cloned into pUC19 with MfeI and KpnI.
3. mEGFP (monomer EGFP) was obtained by two-steps PCR. The point-mutated segments of mEGFP were amplified from plasmid carrying EGFP by KOD polymerase PCR, using primers designed with favored point-mutation sequences. The second PCR produced complete mEGFP by using purified PCR products from former PCR as the templates.
4. Acquire suitable transfection conditions of HEK293 cells by testing with calcium phosphate transfection.
5. The pEGFP-LC3 plasmid had already been tested through transfection
6. DKK-1 promoter and secreting peptide attached reporter gene had also been cloned into pUC19.

week12

FRET SYSTEM

1. transform BL21 with His-tag post 50, His-tag post 75, His-tag post 90
2. transform DH5-a with Plasmid2(DKK1promoter+ALB+mCherry+BGA)
3. transform BL21 with CyPet post+E1/E3/E1E2/E3E4/H7, YPet post + E3/E3E4/G5/H7, pre+CyPet, pre+YPet
4. tranform BL21 with CyPet post + G5 0915, CyPet post + G5 0914, CyPet pre 0915, CyPet pre 0914, YPet pre 0915, YPet pre 0914
5. CyPet post and E1/E3/E1E2/E3E4/H7 were ligated together.
6. YPet post and E3/E3E4/G5/H7 were ligated together.
7. pre and CyPet were ligated together.
8. pre and YPet were ligated together.
9. G5 and CyPet were ligated together.

CELL SYSTEM

1. The first plasmid, DKK1 promoter + secreting mCherry + BGA + pUC19 (plasmid 1s), was constructed.
2. DKK1 promoter + mCherry + BGA + pUC19 (plasmid 1) was also constructed.
3. 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.
4. Adjusting the transfection conditions in DP cells.

week13

FRET SYSTEM

1. CyPet-G5 post were digest checked with restriction enzymes(PstI).
2. CyPet b, SUMO1, YPet j, Ubc9, YPet pre, CyPet pre were digested with restriction enzymes (KpnI&BamHI).
3. CyPet b were ligated into SUMO1.
4. YPet j were ligated into Ubc9.
5. VHH(G5 &H7) were ligated into new skel.
6. KOD polymerase PCR amplification of YPet pre, SUMO1, Ubc9, YPet pre, CyPet pre.

CELL SYSTEM

1. Two plasmids, CMV promoter + mCherry + BGA + pUC19 and CMV promoter + ALB + mCherry + BGA +pUC19, were constructed for observation of transfection effectiveness.
2. The ligation of mEGFP and secreting mEGFP with pUC19 failed.
3. During the testing experiments, most of the cells (including DP cells and HEK293) was CONTIMANATED by other users. We thawed frozen cells and transferred our experimental position to other cell culture room.
4. We got HEK293T cell line from other laboratory and continued our testing experiments.

OCTOBER

week14

FRET SYSTEM

1. CyPet-E3E4 post, YPet-E1E2 post protein expresses in E.coli was checked using a Spectrophotometer.
2. KOD polymerase PCR amplification of YPet-Ubc9 post, CyPet-SUMO1post.

CELL SYSTEM

1. mEGFP and secreting mEGFP were ligated with BGA and cloned into pUC19 with with SalI and HindIII and by two in one ligation.
2. CMV promoter + secreting mEGFP + BGA + pUC19 (plasmid 2s) was constructed.
3. All plasmids had already been constructed.
   1. Plasmid 1: DKK-1 promoter+ mCherry+ BGA
   2. Plasmid 1s: DKK-1 promoter+ ALB+ mCherry+ BGA
   3. Plasmid 2: CMV+ mEGFP+ BGA
   4. Plasmid 2s: CMV+ ALB+ mEGFP+ BGA
   5. Plasmid 3: CMV+ mCherry+ BGA
   6. Plasmid 3s: CMV+ ALB+ mCherry+ BGA
4. We tested plasmid 1 versus plasmid 1s, plasmid 2 versus plasmid 2s, respectively in culture dishes.
5. We concentrated our supernatant in order to amplify our secreted form mCherry for fluorescent detection.
6. We tested the correlation between mCherry expression in HEK293T cell and the varying concentration of sex-hormone (dihydrotestosterone (DHT) and testosterone) at different time point.
7. We also tried different transfection kits to improve our cell’s transfection rate.

week15

CELL SYSTEM

1. The submitted parts, mEGFP, secreting mCherry, and secreting mEGFP were cloned into pSB1C3 respectively.
2. mEGFP and secreting mEGFP were finally cloned into pUC19 respectively after trying many times.
3. We repeated the previous experiments to get more results.
4. We did HUMAN DKK-1 ELISA to detect HEK293T DKK-1 expression at six different DHT concentration, BUT WE FAILED.
5. We got high transfection rate by using calcium phosphate transfection technique and also co-transfected internal control plasmid with plasmid 1s in HEK293T.

LABNOTE ORIGINAL FILE