Difference between revisions of "Team:NYMU-Taipei/Notebook"

Line 33: Line 33:
  
 
<h2>week2</h2>
 
<h2>week2</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Plasmid pSB1C3(ECFP),pSB1C3(SYFP2) and pSB1C3(YFP) were transformed into competent cell DH5-alpha and BL21.</li>
 
<li>Plasmid pSB1C3(ECFP),pSB1C3(SYFP2) and pSB1C3(YFP) were transformed into competent cell DH5-alpha and BL21.</li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Plasmid pUC19 was transformed into competent cell DH5-alpha.</li>
 
<li>Plasmid pUC19 was transformed into competent cell DH5-alpha.</li>
Line 46: Line 46:
  
 
<h2>week3</h2>
 
<h2>week3</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Plasmid pSB1C3-ECFP/EYFP/SYFP2/CYFP were extracted from competent cell BL21 and checked with restriction digestion(NcoI and EcoRI).</li>
 
<li>Plasmid pSB1C3-ECFP/EYFP/SYFP2/CYFP were extracted from competent cell BL21 and checked with restriction digestion(NcoI and EcoRI).</li>
Line 53: Line 53:
 
<li>ECFP/EYFP/SYFP2 were amplified by PCR and extracted from agarose gel.</li>
 
<li>ECFP/EYFP/SYFP2 were amplified by PCR and extracted from agarose gel.</li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Dkk1 promoter was amplified from HEK293 genomic DNA by KOD and DreamTaq polymerase PCR.</li>
 
<li>Dkk1 promoter was amplified from HEK293 genomic DNA by KOD and DreamTaq polymerase PCR.</li>
Line 61: Line 61:
  
 
<h2>week4</h2>
 
<h2>week4</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li>ECPF/EYFP/SYFP2 were amplified by PCR.</li>
 
<li>ECPF/EYFP/SYFP2 were amplified by PCR.</li>
Line 71: Line 71:
  
 
<h2>week5</h2>
 
<h2>week5</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li>CyPet/YPet/ ECFP/ EYFP/SYFP2 inserted intoplasmid pET32a.</li>
 
<li>CyPet/YPet/ ECFP/ EYFP/SYFP2 inserted intoplasmid pET32a.</li>
Line 79: Line 79:
 
<li>Plasmid Fluorescence Protein+pET32a was checked with restriction digestion(BamHI and SalI).</li>
 
<li>Plasmid Fluorescence Protein+pET32a was checked with restriction digestion(BamHI and SalI).</li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>KOD and DreamTaq polymerase PCR amplification of Dkk1 promoter from HEK293 genomic DNA. </li>
 
<li>KOD and DreamTaq polymerase PCR amplification of Dkk1 promoter from HEK293 genomic DNA. </li>
Line 97: Line 97:
  
 
<h2>week6</h2>
 
<h2>week6</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li>CyPet, YPet/ ECFP/ EYFP/SYFP2 post and plasmid pET32a inserted into plasmid pET32a.</li>
 
<li>CyPet, YPet/ ECFP/ EYFP/SYFP2 post and plasmid pET32a inserted into plasmid pET32a.</li>
Line 109: Line 109:
 
<li>The plasmid with LRP6 was extracted from competent cell BL21.</li>
 
<li>The plasmid with LRP6 was extracted from competent cell BL21.</li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>DKK1 promoter junction PCR was conducted.</li>
 
<li>DKK1 promoter junction PCR was conducted.</li>
Line 120: Line 120:
  
 
<h2>week7</h2>
 
<h2>week7</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 128: Line 128:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>The ligation of Plasmid pUC19 and the DKK1 promoter was checked with restriction digestion(EcoRI and XbaI).</li>
 
<li>The ligation of Plasmid pUC19 and the DKK1 promoter was checked with restriction digestion(EcoRI and XbaI).</li>
Line 135: Line 135:
  
 
<h2>week8</h2>
 
<h2>week8</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 143: Line 143:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Preparation for most of the materials including HEK293 (an immortalized kidney cancer cell), medium, and cultural dishes.</li>
 
<li>Preparation for most of the materials including HEK293 (an immortalized kidney cancer cell), medium, and cultural dishes.</li>
Line 152: Line 152:
  
 
<h2>week9</h2>
 
<h2>week9</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 160: Line 160:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Plasmid EGFP-LC3 was extracted from DH5-alpha.</li>
 
<li>Plasmid EGFP-LC3 was extracted from DH5-alpha.</li>
Line 178: Line 178:
  
 
<h2>week10</h2>
 
<h2>week10</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 186: Line 186:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>Reception of immortalized human hair follicle dermal papilla cells and testosterone from our generous collaborator - Dr. Chen Chih-Chiang.</li>
 
<li>Reception of immortalized human hair follicle dermal papilla cells and testosterone from our generous collaborator - Dr. Chen Chih-Chiang.</li>
Line 194: Line 194:
  
 
<h2>week11</h2>
 
<h2>week11</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 202: Line 202:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li> Acquire suitable transfection conditions of HEK293 cells by testing with calcium phosphate transfection.</li>
 
<li> Acquire suitable transfection conditions of HEK293 cells by testing with calcium phosphate transfection.</li>
Line 210: Line 210:
  
 
<h2>week12</h2>
 
<h2>week12</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 218: Line 218:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li>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.</li>
 
<li>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.</li>
Line 228: Line 228:
  
 
<h2>week13</h2>
 
<h2>week13</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 236: Line 236:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
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<h2>week14</h2>
 
<h2>week14</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 262: Line 262:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
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<h2>week15</h2>
 
<h2>week15</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 280: Line 280:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 290: Line 290:
  
 
<h2>week16</h2>
 
<h2>week16</h2>
<p>FRET MODEL</p>
+
<p>FRET SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>
Line 298: Line 298:
 
<li></li>
 
<li></li>
 
</ol>
 
</ol>
<p>CELL MODEL</p>
+
<p>CELL SYSTEM</p>
 
<ol>
 
<ol>
 
<li></li>
 
<li></li>

Revision as of 09:28, 15 October 2018




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 intoplasmid 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

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

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

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

CELL SYSTEM

  1. Reception of immortalized human hair follicle dermal papilla cells and testosterone from our generous collaborator - Dr. Chen Chih-Chiang.
  2. Test the properties of the DP cells before the upcoming transfection experiments.
  3. Test various transfection conditions by using HEK293 cells and PD cells.

week11

FRET SYSTEM

CELL SYSTEM

  1. Acquire suitable transfection conditions of HEK293 cells by testing with calcium phosphate transfection.
  2. The pEGFP-LC3 plasmid had already been tested through transfection
  3. DKK-1 promoter and secreting peptide attached reporter gene had also been cloned into pUC19.

week12

FRET SYSTEM

CELL SYSTEM

  1. 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.
  2. 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