Team:Hong Kong HKUST/notebook/mod2

From 2013.igem.org

(Difference between revisions)
Line 301: Line 301:
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
       <div class='content' align="left">
       <div class='content' align="left">
-
<br><b>June 24 </b><br>
+
June 24 <br>
Wet lab <br>
Wet lab <br>
·      Inoculation of pBlueScript KS(+) for training <br>
·      Inoculation of pBlueScript KS(+) for training <br>
Line 310: Line 310:
Protocols review <br>
Protocols review <br>
<br>
<br>
-
<b>June 25</b> <br>
+
June 25 <br>
Wet lab <br>
Wet lab <br>
·  Plasmid extraction of pBlueScript KS(+)  <br>
·  Plasmid extraction of pBlueScript KS(+)  <br>
Line 318: Line 318:
·      Informal meeting <br>
·      Informal meeting <br>
<br>
<br>
-
<b>June 26</b> <br>
+
June 26 <br>
Wet lab <br>
Wet lab <br>
·      Extraction of gDNA from HepG2 Cells for FABP1 and PPARa <br>
·      Extraction of gDNA from HepG2 Cells for FABP1 and PPARa <br>
-
·      Transformation of pEGFP-N1 <br>
+
·      Transformation of pEGFP-N1 and BBa_K817002 (pFadBA) <br>
-
·      Transformation of BBa_K817002 (pFadBA) <br>
+
<br>
<br>
-
<b>June 27</b> <br>
+
June 27 <br>
Wet lab <br>
Wet lab <br>
-
·      Inoculation of pEGFP-N1 for plasmid extraction <br>
+
·      Inoculation of pEGFP-N1 and BBa_K817002 (pFadBA) for plasmid extraction <br>
-
·      Inoculation of BBa_K817002 (pFadBA) for plasmid extraction <br>
+
<br>
<br>
-
<b>June 28 </b><br>
+
June 28 <br>
Wet lab <br>
Wet lab <br>
-
·      Extraction of pEGFP-N1 plasmids by miniprep <br>
+
·      Extraction of pEGFP-N1 and BBa_K817002 (pFadBA) plasmids by miniprep <br>
-
·      Extraction of BBa_K817002 (pFadBA) <br>
+
·      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 <br>
·      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 <br>
·      Restriction of c(pFadBA) by EcoR1 and Pst1 <br>
·      Restriction of c(pFadBA) by EcoR1 and Pst1 <br>
-
·      Ran 0.8% gel for pEGFP-N1 <br>
+
·      Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (pFadBA) <br>
-
·      Ran 2% gel for BBa_K817002 (pFadBA) <br>
+
<br>
<br>
-
<b>June 30</b> <br>
+
June 30 <br>
Dry lab <br>
Dry lab <br>
-
Experiment planning and protocols revision for next week work<br></br>
+
Experiment planning and protocols revision for next week work <br><br>
-
 
+
       </div>
       </div>
       </div>
       </div>
Line 391: Line 386:
   <div>
   <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br> <b> July 8</b> <br>
 +
Wet lab<br>
 +
·      gDNA extraction from HepG2 cells<br>
 +
·      restriction of BBa_K817002 pFAdBA<br>
 +
·      Gel check for gDNA extraction<br>
 +
·      Inoculation of BBa_J176171<br>
 +
·      Inoculation of BBa_K817002  pFadBA Inoculation of pEGFP-N1 for plasmid extraction<br>
 +
·      New primers for PPARa and FABP1 arrival<br>
 +
·      Ran gel for BBa_K817002 pFadBA restriction check, gel extraction and purification<br>
 +
 
 +
<br> <b> July 9</b> <br>
 +
Wet lab<br>
 +
·      Extraction of BBa_J176171 by miniprep<br>
 +
·      Extraction of BBa_K817002 pFadBA by miniprep<br>
 +
·      Extraction of pEGFP-N1 by miniprep<br>
 +
·      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<br>
 +
·      Restriction of BBa_J176171 for pFadBA Ase1 and BamH1; FABP1 by Xba1 and Not1<br>
 +
·      Ran gel for pEGFP-N1 restriction products<br>
 +
·      Ran gel for BBa_J176171 restriction products<br>
 +
·      PCR PPARa and FABP1 promoters cloning from gDNA<br>
 +
·      BBa_J52034 (FADR) transformation<br>
 +
 
 +
<br> <b> July 10</b> <br>
 +
Wet lab<br>
 +
·      Ran gel for PCR check of PPARa and FAPB1<br>
 +
·      PCR for FABP1, PCR replication<br>
 +
·      BBa_J176171 vector dephosphorylation  by antartic phosphatase<br>
 +
·      Ligation of BBa_K817002 and pFadBA and BBa_J176171 <br>
 +
·      BBa_J52034 (FADR) inoculation<br>
 +
·      Digestion for BBa_K817002 pFadBA extraction<br>
 +
Gel check for BBa_K817002 pFadBA extraction<br>
 +
 
 +
<br> <b> July 11</b> <br>
 +
Wet lab<br>
 +
·      Gel purification for BBa_K817002 pFadBA extraction<br>
 +
·      Gel check for FABP1 PCR product<br>
 +
·      PCR clean-up<br>
 +
·      BBa_J52034 (FADR) miniprep<br>
 +
·      BBa_J52034 restriction by Pst1 HF and Not1 HF<br>
 +
·      PCR  for PPARa<br>
 +
Dry lab<br>
 +
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions<br>
 +
 
 +
<br> <b> July 12</b> <br>
 +
Wet lab<br>
 +
·  Digestion of FABP1 PCR product<br>
 +
·      Ran gel for PCR check of PPARa<br>
 +
·      Digest pEGFP-n1 for BBa_J52034 FADR<br>
 +
·      Ran gel for pEGFP-n1 for BBa_J52034 FADR followed by gel purification<br>
 +
·      BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 and FADR<br>
 +
Dry Lab<br>
 +
Primers design for pCMV cloning for FADR expression<br><br>
 +
 
       </div>
       </div>
   </div>
   </div>

Revision as of 14:56, 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

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 promoter cloning from gDNA
· PCR for 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 promoter cloning from gDNA
· PCR for FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPARa
· Ran gel for PCR chack of 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
· Inoculation of BBa_K817002 pFadBA Inoculation of 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 by miniprep
· Extraction of BBa_K817002 pFadBA by miniprep
· Extraction of 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 restriction products
· Ran gel for 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
Content 1
Week 4
Content 1
Week 5
Content 1

August 2013

Week 1
Content 1
Week 2
Content 1
Week 3
Content 1
Week 4
Content 1

September 2013

Week 1
Content 1
Week 2
Content 1
Week 3
Content 1
Week 4
Content 1
Week 5
Content 1