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Latest revision as of 23:14, 27 September 2013

Protein Trafficking Module's Notebook

June 2013

Week 4

24-6-2013
Inoculation of pCMV/myc/mito

25-6-2013
Plasmid extraction for pCMV/myc/mito
Digestion of pCMV plasmid extraction product with PstI-HF and NotI-HF
Gel electrophoresis of pCMV digested product
Gel check for pCMV digestion product

26-6-2013
Gel purification of pCMV gel product
Gel check of pCMV gel purified product
PCR clone MLS from pCMV/myc/mito
Transformation of pEGFP-N1 with SURE

27-6-2013
Gel check for the MLS PCR product and PCR Clean Up
Digestion of MLS clean up product with EcoRI & PstI-HF
Inoculation of pEGFP-N1

28-6-2013
PCR Clean up of digested MLS
Plasmid Extraction of pEGFP-N1
Digestion of pEGFP-N1 with PstI-HF & NotI-HF

July 2013

Week 1

02-07-2013
Gel Check of pEGFP-N1 plasmid extraction product to confirm presence of DNA
Digestion of pEGFP-N1 (with NotI-HF and PstI-HF) 2nd EGFP product:repeat digestion
Inoculation of pEGFP-N1 3rd EGFP: repeat Inoculation
Get pSB1C3.J04450 plasmid extraction product from Bida
Digestion of pSB1C3 with EcoRI and PstI-HF

03-07-2013
Gel electrophoresis of digested EGFP (2nd)
Plasmid extraction of pEGFP-N1 (3rd)
Digetion of pEGFP-N1 (3rd) using NotI-HF and PstI-HF
Ligation of MLS with pSB1C3
Transformation of ligated pSB1C3-MLS

04-07-2013
Gel purification of pEGFP-N1 (2nd) (cont.)
Gel analysis and gel electrophoresis of pEGFP-N1 (3rd)
Run gel for remaining 3rd EGFP (digested)
Ethanol precipitation of pEGFP-N1 (4th) (using remaining 3rd EGFP plasmid)
Digestion of pEGFP-N1 (4th) and gel electrophoresis
Transformation of pEGFP-N1 into DH10B
Colony PCR of MLS biobrick and gel electrophoresis
Inoculation of MLS colony no.2 & spread the remaining re-suspend colony 2 on CAM plate

05-07-2013
pEGFP-N1 troubleshoot
Digestion by PstI-HF, NotI-HF, PstI-HF +NotI-HF and gel check
Inoculate EGFP from transformed new competent cell
Plasmid extraction of MLS Biobrick
Sanger sequencing of MLS

Week 2

08-07-2013
Result of MLS Sanger sequencing showed that DNA is not enough, the sequencing failed
Inoculation of MLS.pSB1C3
Plasmid extraction of pEGFP-N1
Digestion of pEGFP-N1 with NotI-HF & PstI-HF
Run gel, and it turned out that NotI-HF did not work

09-07-2013
Plasmid extraction of MLS biobrick
Digestion check on MLS.pSB1C3 product with EcoRI & PstI
Sanger sequencing of MLS biobrick
pEGFP-N1 digestion with PstI & NotI

10-07-2013
pCMV/myc/mito & pEGFP-N1 ligation, and transform into SURE on AMP plate
MLS biobrick Sanger sequencing result showed there is no pellet, repeat Sanger sequencing
Run gel for digestion check of MLS.pSB1C3 with EcoRI & PstI

11-07-2013
Inoculation of pCMV/myc/mito.EGFP
MLS biobrick Sanger sequencing (Cont.)

12-07-2013
Plasmid extraction of pCMV/myc/mito.EGFP
pCMV/myc/mito.EGFP transformation check by digesting with PstI & NotI

Spread plate for confirmed colony

Week 3

15/7/2013
pCMV/myc/mito.GFP inoculation

16/7/2013
Plasmid Extraction of pCMV/myc/mito.GFP
Nanodrop: concentration is not high enough, will repeat inoculation

17-7-2013
Inoculate 4 tubes of pCMV/myc/mito.GFP

18-7-2013
pCMV/myc/mito.GFP plasmid extraction
PCR clone MLS (RFC25)

19-7-2013
Run gel for MLS(RFC25) PCR product, PCR clean up
Digestion of the MLS PCR product using EcoRI and PstI, gel check
Digestion of pSB1C3 with EcoRI & PstI, run gel

Week 4

22-7-2013
Gel purification of pSB1C3
Gel check of pSB1C3: band is too pale, repeat inoculation
Inoculation of pSB1C3.J04450
Repeat PCR of MLS(RFC25)
Gel check of PCR product and PCR clean up
Digestion of MLS(RFC25) with EcoRI & SpeI, run gel

23-7-2013
MLS(RFC25) Gel purification (Cont), and gel check
pSB1C3 plasmid extraction
Digestion of pSB1C3 with EcoRI & SpeI

24-7-2013
pSB1C3 Gel purification (Cont.)
MLS(RFC25) & pSB1C3 ligation, and transform to CAM plate

25-7-2013
Colony PCR of MLS.pSB1C3(RFC25)
Inoculation of MLS.pSB1C3(RFC25)

26-7-2013
Plasmid extraction of MLS.pSB1C3(RFC25)
Digestion of ligated MLS-pSB1C3 with EcoRI & SpeI, gel check
Gel check for MLS.pSB1C3(RFC25) colony PCR product
Spread the remaining re-suspend of correct colony on CAM plate

Week 5

29-7-2013
Repeat digestion of pSB1C3.MLS(RFC25) with EcoRI and SpeI, gel check and Sanger sequence
Spread plate of GFP(RFC25) transformed colonies

30-7-2013
pSB1C3.MLS (RFC25) digestion with EcoRI and SpeI
Gel electrophoresis of digested pSB1C3.MLS product
Streak plate for pSB1C3.MLS colony 1,2, and 3
Sanger sequencing of pSB1C3.MLS
Streak plate of EGFP(RFC25) colonies

31-7-2013
pSB1C3.MLS (RFC25) Sanger sequence(Cont.)
GFP (RFC25) inoculation

01-08-2013
GFP(RFC25) plasmid extraction
Prepare the glycerol stock and stored it
Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis

02-08-2013
GFP (RFC25) plasmid extraction
Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis
Inoculate BBa_J176171 using Amp LB, and BBa_K404108 using CAM LB

August 2013

Week 1

5-8-2013
GFP (RFC25) PCR Check with VR VF2, gel check (over ran)
BBa_J176171 digestion using SpeI and PstI-HF to extract the backbone
PolyA / BBa_K404108 digestion using XbaI and PstI-HF
Inoculation of pSB1C3

6-8-2013
Repeat PCR check of GFP (RFC25)
pSB1C3 plasmid extraction
PolyA / BBa_K404108 gel purification and gel check
BBa_J176171 PCR clean up
BBa_K929000 & BBa_J52034 transformation using CAM plate
PCR amplify MLS in RFC 10 and RFC25 format

7-8-2013
MLS (RFC 10 & RFC25) PCR clean up
Nanodrop and gel check for the PCR clean up product
Digestion using EcoRI and PstI
PolyA / BBa_K404108 gel check
pSB1C3 digestion with EcoRI & PstI
GFP(RFC25) digestion with EcoRI & SpeI

8/8/2013
Gel purification of GFP(RFC25) & pSB1C3, then nanodrop and gel check
polyA digestion with EcoRI and XbaI, and PCR clean up
Inoculation of BBa_K929000 & BBa_J52034

9/8/2013
BBa_K929000 & BBa_J52034 plasmid extraction: J52034 is not transformed
MLS.pSB1C3 & GFP.PolyA ligation, and transform to SURE on CAM plate

Week 2

12/8/2013

Digestion check of K929000 with ApaLI & NdeI
MLS.pSB1C3 & GFP.PolyA inoculation
Transformation of J52034

13/08/2013
MLS(RFC10).pSB1C3 & gpf.polyA plasmid extraction
Digestion of MLS(RFC10).pSB1C3 & gpf.polyA with EcoRI-HF and PstI-HF
K929000 gel check
pSB1C3 digestion with EcoRI & PstI
MLS(RFC25).pSB1C3
Streak plate with transformed MLS(RFC25).pSB1C3 to purify colony

14/08/2013
Inoculation of MLS(RFC25).pSB1C3 & GFP.PolyA

15/08/2013
pSB1C3 gel purification
MLS(RFC10).pSB1C3 Sanger sequencing (did not submit as the colony turn out to be not single)
Plasmid extraction of MLS(RFC25).pSB1C3 & GFP
Digestion check of MLS(RFC25) with EcoRI & PstI; GFP.polya with ApaLI & AgeI HF

16/08/2013
pCMV (Amplify by PCR from pEGFP-N1) PCR clean up
pSB1C3 dephosphorylation
Run gel of MLS(RFC25).pSB1C3 & gpf.polya
MLS(RFC25).pSB1C3 PCR check
Streak plate with transformed MLS(RFC10).pSB1C3 to get single colony
Inoculation of pEGFP-N1, pCMV/my/mito.GFP, pCMV/myc/mito
Streak plate with transformed pEGFP-N1

Week 3

19/08/2013
Ligation of CMV and pSB1C3 (1st try)
Transformation of CMV and pSB1C3 into DH5alpha
Streak pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP to AMP plate
MLS(RFC25).pSB1C3 & GFP.polya digestion with AgeI & ApaLI, and transformation of the correct colony
MLS(RFC25).pSB1C3 Sanger Sequencing

20/8/2013
MLS(RFC25).pSB1C3 Sanger Sequencing(Cont.)
Plasmid extraction of pEGFP-N1, pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP
MLS(RFC25).pSB1C3 digestion with AgeI-HF & PstI-HF
GFP.polya.pSB1C3(1st try) digestion with NgoMIV & PstI-HF
Repeat PCR for CMV(2nd try), digestion with EcoRI-HF & PstI-HF, PCR clean up

21/08/2013
Gel purification of GFP/polyA digested with NgoMIV & PstI-HF : too low concentration
EtOH precipitation of GFP/polyA digested with NgoMIV & PstI-HF
Pcmv.pSB1C3(2nd) ligation, and transformation to DH5alpha
MLS(RFC10) bacteria inoculation

22/08/2013
MLS(RFC10).pSB1C3 plasmid extraction, PCR check with VR VF2
Repeat digestion of GFP.polyA with NgoMIV & PstI-HF (2nd) with NgoMIV & PstI-HF
Pcmv.pSB1C3 (3rd) ligation and transformation
Inoculation of Pcmv.pSB1C3 (2nd)
GFP.polyA with NgoMIV & PstI-HF (1st) gel check

23/08/2013
Sanger Sequencing for MLS(RFC10).pSB1C3 and MLS(RFC25).pSB1C3
Pcmv.pSB1C3 (2nd) plasmid extraction, and digestion check with EcoRI & NdeI
Streak the right colony of Pcmv.pSB1C3 (2nd) into plate
GFP.polyA with NgoMIV & PstI-HF (2nd) gel purification

Week 4

26/8/2013
Sanger sequencing for Pcmv.pSB1C3 (#1), MLS(RFC10).pSB1C3 (#4.3), and MLS(RFC25).pSB1C3 #1.1)
Gel check for GFP.polyA (NgoMIV & PstI-HF) for 1st & 2nd
GFP.polyA digestion with XbaI & PstI-HF, run gel and gel purification
Pcmv digestion with SpeI & PstI-HF, dephosphorylated
MLS(RFC25).pSB1C3 digestion with AgeI & PstI-HF, dephosphorylated
Repeat Sequencing for MLS(RFC25).pSB1C3

27/8/2013
MLS(RFC25).pSB1C3 Sequencing (Cont.)
Repeat Sequencing for Pcmv.pSB1C3
MLS(RFC10).pSB1C3 Sequencing (Cont.), transformed new plate with correct colony (#4.3)
GFP.polyA(XbaI & PstI-HF) gel Purification(Cont.), and gel check
Gel check for Pcmv (SpeI & PstI-HF) & MLS(RFC25).pSB1C3(AgeI & PstI-HF)
GFP(RFC25) digestion with EcoRI-HF & PstI-HF, transformed new plate for correct colony (#2)

28/8/2013
Ligation of Pcmv (S&P) and GFP (X&P), transformed
Ligation of MLS (A&P) and GFP (N&P), transformed
Pcmv.pSB1C3 and MLS(RFC25) sequence confirmed
GFP(RFC25) gel purification, EtOH precipitation
GFP(RFC25) nanodrop: concentration is too low, abandone

29/8/2013
Inoculation of MLS.GFP.polyA and CMV.GFP.polyA

30/8/2013
Plasmid extraction of MLS.GFP.polyA & CMV.GFP.polyA
Digestion check of MLS.GFP.polyA with EcoRV-HF & NdeI, and CMV.GFP.polyA with EcoRV-HF & AgeI-HF
Transfection of CMV.GFP.polyA #1

September 2013

Week 1

2/9/2013
Repeat Digestion check for MLS.GFP.polyA & CMV.GFP.polyA
Gel check of CMV.GFP.polyA: long gel, delayed since not enough time to run gel
Digestion of MLS.GFP.polyA with XbaI & PstI-HF

3/9/2013
Digestion check of CMV.GFP.polyA with EcoRV-HF & AgeI-HF
Run gel and gel purification of MLS.GFP.polyA (X & P)
Pcmv digestion with SpeI and PstI-HF, dephosphorylated and PCR clean up
BBa_J176171 digestion with SpeI & PstI-HF, dephosphorylation and PCR clean up

4/9/2013
GEl purification for MLS.GFP.polyA

5/9/2013
Gel check for CMV.GFP.polyA (E&P), CMV.pSB1C3 (E&P), and GFP.polyA.pSB1C3(E&P)
Run gel for MLS.GFP.polyA(XbaI&PstI-HF), CMV(SpeI&PstI-HF), BBa_J176171(SpeI&PstI-HF), and CMV.GFP.polyA (EcoRV-HF, AgeI-HF)

6/9/2013
Digestion of CMV.pSB1C3 (1) & GFP.polyA(4) by EcoRI-HF & PstI-HF
Gel check: decided that cmv is defect, will order new primer

Week 2

9/9/2013
pSB1C3 digestion with EcoRI-HF & PstI-HF
Inoculation of pEGFP-n1 (from confirmed colony spread plate)

10/9/2013
Gel purification of pSB1C3
Plasmid extraction of pEGFP-N1

12/9/2013
Digestion of MLS.GFP.polyA and GFP.polyA with SpeI & PstI, dephosphorylated
Amplify CMV with PCR using new primer

Week 3

Content 1

Week 4

Content 1

Week 5

Content 1

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@@ Line 238: / Line 269: @@
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+ <a href="https://2013.igem.org/Main_Page"><img id="iGEM_Logo" src="https://static.igem.org/mediawiki/2013/4/46/Igem_qgem_logo.png"></a>
+<a href="http://www.ust.hk/eng/index.htm"><img id="hkust_Logo" src="https://static.igem.org/mediawiki/2013/5/55/Hkust_logo.gif"></a>
+<ol class="pos_fixed">
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1">Module 1</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod2">Module 2</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod3">Module 3</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod4">Module 4</a></il>
+</ol>
 <a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
 <div id="cover"></div>
@@ Line 254: / Line 315: @@
 <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
 <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/attribution">Attribution</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/acknowledge">Acknowledgement</a></li>
 </ul>
 </li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/project">Project</a>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project">Project</a>
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 <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modelling">Modelling</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/data">Data Page</a></li>
 <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/datapage">Data Page</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Results</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Result</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/future">Future Work</a></li>
 </ul>
 </li>
@@ Line 270: / Line 334: @@
 <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
 <ul>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">notebook</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">protocols</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">safety</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li>
 </ul>
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@@ Line 285: / Line 350: @@
 <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
 <ul>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Interviews">Interviews</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/cp">Country Profile</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Presentation">Presentation</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/blog">Blog</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Videos">Videos</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/interview">Interviews</a></li>
-<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Article">Article</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/article/genet">Article</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/video">Videos</a></li>
+<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/presentation">Presentations</a></li>
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-<br><br><br><br><br><br><br>
+<br><br><br><br><br><br><br><br>
+<div id="title"><center><h3>Protein Trafficking Module's Notebook</h3></center></div>
 <div id="flight">
   <div id="satu" align="center"> <h1>June 2013</h1>
@@ Line 300: / Line 369: @@
        <div>
        <a href='#' class='head'>Week 4</a>
-       <div class='content' align="left">Wet lab<br>
+       <div class='content' align="left">
-·      Inoculation of pBlueScript KS(+) for training<br>
+<p>&nbsp;</p>
-·      Autoclave basic materials<Br>
+<p><strong>24-6-2013</strong> <br />
-·      Preparing LB<br>
+  Inoculation of pCMV/myc/mito </p>
-·      LB-Ampicillin plates poured<br>
+<p>&nbsp;</p>
-Dry lab<br>
+<p><strong>25-6-2013</strong> <br />
-Protocols review
+  Plasmid extraction for pCMV/myc/mito <br />
+  Digestion of pCMV plasmid extraction product with PstI-HF and NotI-HF <br />
+  Gel electrophoresis of pCMV digested product <br />
+Gel check for pCMV digestion product </p>
+<p>&nbsp;</p>
+<p><strong>26-6-2013</strong> <br />
+  Gel purification of pCMV gel product <br />
+  Gel check of pCMV gel purified product <br />
+  PCR clone MLS from pCMV/myc/mito <br />
+Transformation of pEGFP-N1 with SURE </p>
+<p>&nbsp;</p>
+<p><strong>27-6-2013</strong> <br />
+  Gel check for the MLS PCR product and PCR Clean Up <br />
+  Digestion of MLS clean up product with EcoRI &amp; PstI-HF <br />
+Inoculation of pEGFP-N1 </p>
+<p>&nbsp;</p>
+<p><strong>28-6-2013</strong> <br />
+  PCR Clean up of digested MLS <br />
+  Plasmid Extraction of pEGFP-N1 <br />
+Digestion of pEGFP-N1 with PstI-HF  &amp; NotI-HF </p>
+<p>&nbsp;</p>
        </div>
        </div>
@@ Line 316: / Line 405: @@
     <div>
        <a href='#' class='head'>Week 1</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>02-07-2013</strong> <br />
+  Gel Check of pEGFP-N1 plasmid extraction product to confirm presence of DNA <br />
+  Digestion of pEGFP-N1 (with NotI-HF and PstI-HF) 2nd EGFP product:repeat digestion <br />
+  Inoculation of pEGFP-N1 3rd EGFP: repeat Inoculation <br />
+  Get pSB1C3.J04450 plasmid extraction product from Bida <br />
+  Digestion of pSB1C3 with EcoRI and PstI-HF </p>
+<p>&nbsp;</p>
+<p><strong>03-07-2013</strong> <br />
+  Gel electrophoresis of digested EGFP (2nd) <br />
+  Plasmid extraction of pEGFP-N1 (3rd) <br />
+  Digetion of pEGFP-N1 (3rd) using NotI-HF and PstI-HF <br />
+  Ligation of MLS with pSB1C3 <br />
+Transformation of ligated pSB1C3-MLS </p>
+<p>&nbsp;</p>
+<p><strong>04-07-2013</strong> <br />
+  Gel purification of pEGFP-N1 (2nd) (cont.) <br />
+  Gel analysis and gel electrophoresis of pEGFP-N1 (3rd) <br />
+  Run gel for remaining 3rd EGFP (digested) <br />
+  Ethanol precipitation of pEGFP-N1 (4th) (using remaining 3rd EGFP plasmid) <br />
+  Digestion of pEGFP-N1 (4th) and gel electrophoresis <br />
+  Transformation of pEGFP-N1 into DH10B <br />
+  Colony PCR of MLS biobrick and gel electrophoresis <br />
+Inoculation of MLS colony no.2 &amp; spread the remaining re-suspend colony 2 on CAM plate </p>
+<p>&nbsp;</p>
+<p><strong>05-07-2013</strong> <br />
+  pEGFP-N1 troubleshoot <br />
+  Digestion by PstI-HF, NotI-HF, PstI-HF +NotI-HF and gel check <br />
+  Inoculate EGFP from transformed new competent cell <br />
+  Plasmid extraction of MLS Biobrick <br />
+Sanger sequencing of MLS </p>
+<p>&nbsp;</p>
        </div>
     </div>
@@ Line 322: / Line 443: @@
     <div>
        <a href='#' class='head'>Week 2</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>08-07-2013</strong> <br />
+  Result of MLS Sanger sequencing showed that DNA is not enough, the sequencing failed <br />
+  Inoculation of MLS.pSB1C3 <br />
+  Plasmid extraction of pEGFP-N1 <br />
+  Digestion of pEGFP-N1 with NotI-HF &amp; PstI-HF <br />
+  Run gel, and it turned out that NotI-HF did not work </p>
+<p>&nbsp;</p>
+<p><strong>09-07-2013</strong> <br />
+  Plasmid extraction of MLS biobrick <br />
+  Digestion check on MLS.pSB1C3 product with EcoRI &amp; PstI <br />
+  Sanger sequencing of MLS biobrick <br />
+pEGFP-N1 digestion with PstI &amp; NotI </p>
+<p>&nbsp;</p>
+<p><strong>10-07-2013</strong> <br />
+  pCMV/myc/mito &amp; pEGFP-N1 ligation, and transform into SURE on AMP plate <br />
+  MLS biobrick Sanger sequencing result showed there is no pellet, repeat Sanger sequencing <br />
+Run gel for digestion check of MLS.pSB1C3 with EcoRI &amp; PstI </p>
+<p>&nbsp;</p>
+<p><strong>11-07-2013</strong> <br />
+  Inoculation of pCMV/myc/mito.EGFP <br />
+MLS biobrick Sanger sequencing (Cont.) </p>
+<p>&nbsp;</p>
+<p><strong>12-07-2013</strong> <br />
+  Plasmid extraction of pCMV/myc/mito.EGFP <br />
+pCMV/myc/mito.EGFP transformation check by digesting with PstI &amp; NotI</p>
+<p>Spread plate for confirmed colony </p>
+<p>&nbsp;</p>
        </div>
     </div>
@@ Line 328: / Line 477: @@
     <div>
        <a href='#' class='head'>Week 3</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>15/7/2013</strong><br />
+  pCMV/myc/mito.GFP inoculation</p>
+<p>&nbsp;</p>
+<p><strong>16/7/2013</strong><br />
+  Plasmid Extraction of pCMV/myc/mito.GFP<br />
+Nanodrop: concentration is not high enough, will repeat inoculation</p>
+<p>&nbsp;</p>
+<p><strong>17-7-2013</strong><br />
+  Inoculate 4 tubes of pCMV/myc/mito.GFP</p>
+<p>&nbsp;</p>
+<p><strong>18-7-2013</strong><br />
+  pCMV/myc/mito.GFP plasmid extraction<br />
+PCR clone MLS (RFC25)</p>
+<p>&nbsp;</p>
+<p><strong>19-7-2013</strong><br />
+  Run gel for MLS(RFC25) PCR product, PCR clean up<br />
+  Digestion of the MLS PCR product using EcoRI and PstI, gel check<br />
+Digestion of pSB1C3 with EcoRI &amp; PstI, run gel</p>
+<p>&nbsp;</p>
        </div>
     </div>
@@ Line 334: / Line 503: @@
     <div>
        <a href='#' class='head'>Week 4</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>22-7-2013</strong><br />
+  Gel purification of pSB1C3<br />
+  Gel check of pSB1C3: band is too pale, repeat inoculation<br />
+  Inoculation of pSB1C3.J04450<br />
+  Repeat PCR of MLS(RFC25)<br />
+  Gel check of PCR product and PCR clean up<br />
+  Digestion of MLS(RFC25) with EcoRI &amp; SpeI, run gel</p>
+<p>&nbsp;</p>
+<p><strong>23-7-2013<br />
+  </strong>MLS(RFC25) Gel purification (Cont), and gel check<br />
+  pSB1C3 plasmid extraction<br />
+Digestion of pSB1C3 with EcoRI &amp; SpeI</p>
+<p>&nbsp;</p>
+<p><strong>24-7-2013</strong><br />
+  pSB1C3 Gel purification (Cont.)<br />
+MLS(RFC25) &amp; pSB1C3 ligation, and transform to CAM plate</p>
+<p>&nbsp;</p>
+<p><strong>25-7-2013</strong><br />
+  Colony PCR of MLS.pSB1C3(RFC25)<br />
+Inoculation of MLS.pSB1C3(RFC25)</p>
+<p>&nbsp;</p>
+<p><strong>26-7-2013</strong><br />
+  Plasmid extraction of MLS.pSB1C3(RFC25)<br />
+  Digestion of ligated MLS-pSB1C3 with EcoRI &amp; SpeI, gel check<br />
+  Gel check for MLS.pSB1C3(RFC25) colony PCR product<br />
+Spread the remaining re-suspend of correct colony on CAM plate</p>
+<p>&nbsp;</p>
        </div>
     </div>
@@ Line 340: / Line 537: @@
     <div>
        <a href='#' class='head'>Week 5</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>29-7-2013</strong><br />
+  Repeat digestion of pSB1C3.MLS(RFC25) with EcoRI and SpeI, gel check and Sanger sequence<br />
+  Spread plate of GFP(RFC25) transformed colonies</p>
+<p>&nbsp;</p>
+<p><strong>30-7-2013</strong><br />
+  pSB1C3.MLS (RFC25) digestion with EcoRI and SpeI<br />
+  Gel electrophoresis of digested pSB1C3.MLS product<br />
+  Streak plate for pSB1C3.MLS colony 1,2, and 3<br />
+  Sanger sequencing of pSB1C3.MLS<br />
+Streak plate of EGFP(RFC25) colonies</p>
+<p>&nbsp;</p>
+<p><strong>31-7-2013</strong><br />
+  pSB1C3.MLS (RFC25) Sanger sequence(Cont.)<br />
+GFP (RFC25) inoculation</p>
+<p>&nbsp;</p>
+<p><strong>01-08-2013</strong><br />
+  GFP(RFC25) plasmid extraction<br />
+  Prepare the glycerol stock and stored it<br />
+Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis</p>
+<p>&nbsp;</p>
+<p><strong>02-08-2013</strong><br />
+  GFP (RFC25) plasmid extraction<br />
+  Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis<br />
+Inoculate <a href="http://parts.igem.org/Part:BBa_J176171">BBa_J176171</a> using Amp LB, and <a href="http://parts.igem.org/Part:BBa_K404108">BBa_K404108</a> using CAM LB</p>
+<p>&nbsp;</p>
        </div>
     </div>
@@ Line 351: / Line 574: @@
        <div class='content' align="left">
 <p>&nbsp;</p>
-<p><strong>August 5</strong></p>
+<p><strong>5-8-2013</strong><br />
-<p>GFP (25)PCR Check with VR VF2</p>
+  GFP (RFC25) PCR Check with VR VF2, gel check (over ran)<br />
-<ul>
+   BBa_J176171 digestion using SpeI and PstI-HF to extract the backbone<br />
-  <blockquote>
+   PolyA / BBa_K404108 digestion using XbaI and PstI-HF<br />
-    <p>- Gel check(overan)</p>
+   Inoculation of pSB1C3</p>
-   </blockquote>
-</ul>
-<p>BBa_J176171</p>
-<ul>
-  <blockquote>
-    <p>- Digestion using SpeI and PstI-HF to extract the backbone</p>
-   </blockquote>
-</ul>
-<p>PolyA / BBa_K404108</p>
-<ul>
-  <blockquote>
-    <p>- Digestion using XbaI and PstI-HF</p>
-    <p>- Gel electrophoresis</p>
-    <p>- Run Gel </p>
-   </blockquote>
-</ul>
-<p>Inoculation of pSB1C3</p>
 <p>&nbsp;</p>
-<p><strong>August 6</strong></p>
+<p><strong>6-8-2013</strong><br />
-<p>GFP (25)</p>
+   Repeat PCR check of GFP (RFC25)<br />
-<ul>
+   pSB1C3 plasmid extraction<br />
-   <blockquote>
+   PolyA / BBa_K404108 gel purification and gel check<br />
-    <p>- Repeat PCR check</p>
+   BBa_J176171 PCR clean up<br />
-    <p>- Run Gel</p>
+   <a href="http://parts.igem.org/Part:BBa_K929000">BBa_K929000</a> &amp; <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> transformation using CAM plate<br />
-   </blockquote>
+PCR amplify MLS in RFC 10 and RFC25 format</p>
-</ul>
-<p>pSB1C3</p>
-<ul>
-  <blockquote>
-    <p>- Plasmid extraction</p>
-   </blockquote>
-</ul>
-<p>PolyA / BBa_K404108</p>
-<ul>
-  <blockquote>
-    <p>- Gel purification</p>
-    <p>- Gel check</p>
-   </blockquote>
-</ul>
-<p>BBa_J176171</p>
-<ul>
-  <blockquote>
-    <p>- PCR clean up</p>
-   </blockquote>
-</ul>
-<p>BBa_K929000 &amp; BBa_J52034 </p>
-<ul>
-  <blockquote>
-    <p>- Transformation using CAM plate</p>
-  </blockquote>
-</ul>
-<p>MLS</p>
-<ul>
-  <blockquote>
-    <p>- PCR amplify MLS in RFC 10 and 25 format</p>
-  </blockquote>
-</ul>
 <p>&nbsp;</p>
-<p><strong>August 7</strong></p>
+<p><strong>7-8-2013</strong><br />
-<p>MLS (RFC 10 &amp; 25)</p>
+  MLS (RFC 10 &amp; RFC25) PCR clean up<br />
-<ul>
+  Nanodrop and gel check for the PCR clean up product<br />
-  <blockquote>
+  Digestion using EcoRI and PstI<br />
-    <p>- PCR clean up</p>
+   PolyA / BBa_K404108 gel check<br />
-    <p>- Nanodrop for the PCR clean up product</p>
+   pSB1C3 digestion with EcoRI &amp; PstI<br />
-    <p>- Gel check for the PCR clean up product</p>
+GFP(RFC25) digestion with EcoRI &amp; SpeI</p>
-    <p>- Digestion using EcoRI and PstI</p>
-   </blockquote>
-</ul>
-<p>PolyA / BBa_K404108</p>
-<ul>
-  <blockquote>
-    <p>- Gel check</p>
-   </blockquote>
-</ul>
-<p>pSB1C3 </p>
-<ul>
-  <blockquote>
-    <p>- Digest with EcoRI &amp; PstI </p>
-    <p>- Run gel and cut gel </p>
-  </blockquote>
-</ul>
-<p>GFP(25) </p>
-<ul>
-  <blockquote>
-    <p>- Digestion with EcoRI &amp; SpeI</p>
-    <p>- Run gel and cut gel </p>
-  </blockquote>
-</ul>
 <p>&nbsp;</p>
-<p><strong>August 8</strong></p>
+<p><strong>8/8/2013</strong><br />
-<p>GFP25 &amp; pSB1C3</p>
+   Gel purification of<strong> </strong>GFP(RFC25) &amp; pSB1C3, then nanodrop and gel check <br />
-<ul>
+   polyA digestion with EcoRI and XbaI, and PCR clean up<br />
-<blockquote>
+Inoculation of BBa_K929000 &amp; BBa_J52034</p>
-   <p>- Gel purification</p>
-    <p>- Nanodrop</p>
-    <p>- Gel check </p>
-   </blockquote>
-</ul>
-<p>polyA</p>
-<ul>
-  <blockquote>
-    <p>- Digestion with EcoRI and XbaI</p>
-    <p>- PCR clean up</p>
-  </blockquote>
-</ul>
-<p>BBa_K929000 &amp; BBa_J52034 </p>
-<ul>
-  <blockquote>
-    <p>- Inoculation </p>
-  </blockquote>
-</ul>
 <p>&nbsp;</p>
-<p><strong>August 9</strong></p>
+<p><strong>9/8/2013</strong><br />
-<p>BBa_K929000 &amp; BBa_J52034 </p>
+  BBa_K929000 &amp; BBa_J52034 plasmid extraction: J52034 is not transformed<br />
-<p>- Plasmid extraction: J52034 is not transformed</p>
+MLS.pSB1C3 &amp; GFP.PolyA ligation, and transform to SURE on CAM plate</p>
-<p>MLS.pSB1C3 &amp; GFP.PolyA</p>
-<p>- Ligation</p>
-<p>- Transformation to SURE on CAM plate</p>
 <p>&nbsp;</p>
        </div>
      </div>
      <div>
        <a href='#' class='head'>Week 2</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>12/8/2013</strong></p>
+<p>Digestion check of K929000 with ApaLI &amp; NdeI<br />
+  MLS.pSB1C3 &amp; GFP.PolyA inoculation<br />
+Transformation of J52034</p>
+<p>&nbsp;</p>
+<p><strong>13/08/2013</strong><br />
+  MLS(RFC10).pSB1C3 &amp; gpf.polyA plasmid extraction<br />
+  Digestion of MLS(RFC10).pSB1C3 &amp; gpf.polyA with EcoRI-HF and PstI-HF <br />
+  K929000 gel check<br />
+  pSB1C3 digestion with EcoRI &amp; PstI<br />
+  MLS(RFC25).pSB1C3<br />
+Streak plate with transformed MLS(RFC25).pSB1C3 to purify colony</p>
+<p>&nbsp;</p>
+<p><strong>14/08/2013</strong><br />
+  Inoculation of MLS(RFC25).pSB1C3 &amp; GFP.PolyA</p>
+<p>&nbsp;</p>
+<p><strong>15/08/2013</strong><br />
+  pSB1C3 gel purification<br />
+  MLS(RFC10).pSB1C3 Sanger sequencing (did not submit as the colony turn out to be not single)<br />
+  Plasmid extraction of MLS(RFC25).pSB1C3 &amp; GFP<br />
+Digestion check of MLS(RFC25) with EcoRI &amp; PstI; GFP.polya with ApaLI &amp; AgeI HF</p>
+<p>&nbsp;</p>
+<p><strong>16/08/2013</strong><br />
+  pCMV (Amplify by PCR from pEGFP-N1) PCR clean up<br />
+  pSB1C3 dephosphorylation<br />
+  Run gel of MLS(RFC25).pSB1C3 &amp; gpf.polya<br />
+  MLS(RFC25).pSB1C3 PCR check<br />
+  Streak plate with transformed MLS(RFC10).pSB1C3 to get single colony <br />
+  Inoculation of pEGFP-N1, pCMV/my/mito.GFP, pCMV/myc/mito<br />
+Streak plate with transformed pEGFP-N1</p>
+<p>&nbsp;</p>
        </div>
      </div>
      <div>
        <a href='#' class='head'>Week 3</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>19/08/2013</strong><br />
+  Ligation of CMV and pSB1C3 (1st try)<br />
+  Transformation of CMV and pSB1C3 into DH5alpha<br />
+  Streak pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP to AMP plate<br />
+  MLS(RFC25).pSB1C3 &amp; GFP.polya digestion with AgeI &amp; ApaLI, and transformation of the correct colony<br />
+  MLS(RFC25).pSB1C3 Sanger Sequencing</p>
+<p>&nbsp;</p>
+<p><strong>20/8/2013</strong><br />
+  MLS(RFC25).pSB1C3 Sanger Sequencing(Cont.)<br />
+  Plasmid extraction of pEGFP-N1, pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP<br />
+  MLS(RFC25).pSB1C3 digestion with AgeI-HF &amp; PstI-HF<br />
+  GFP.polya.pSB1C3(1st try) digestion with NgoMIV &amp; PstI-HF<br />
+Repeat PCR for CMV(2nd try), digestion with EcoRI-HF &amp; PstI-HF, PCR clean up</p>
+<p>&nbsp;</p>
+<p><strong>21/08/2013</strong><br />
+  Gel purification of GFP/polyA digested with NgoMIV &amp; PstI-HF : too low concentration<br />
+  EtOH precipitation of GFP/polyA digested with NgoMIV &amp; PstI-HF<br />
+  P<i>cmv</i>.pSB1C3(2nd) ligation, and transformation to DH5alpha<br />
+MLS(RFC10) bacteria inoculation</p>
+<p>&nbsp;</p>
+<p><strong>22/08/2013</strong><br />
+  MLS(RFC10).pSB1C3 plasmid extraction, PCR check with VR VF2<br />
+  Repeat digestion of GFP.polyA with NgoMIV &amp; PstI-HF (2nd) with NgoMIV &amp; PstI-HF<br />
+  P<i>cmv</i>.pSB1C3 (3rd) ligation and transformation<br />
+  Inoculation of P<i>cmv</i>.pSB1C3 (2nd)<br />
+  GFP.polyA with NgoMIV &amp; PstI-HF (1st) gel check<br />
+</p>
+<p><br />
+  <strong>23/08/2013</strong><br />
+  Sanger Sequencing for MLS(RFC10).pSB1C3 and MLS(RFC25).pSB1C3<br />
+  P<i>cmv</i>.pSB1C3 (2nd) plasmid extraction, and digestion check with EcoRI &amp; NdeI<br />
+  Streak the right colony of P<i>cmv</i>.pSB1C3 (2nd) into plate <br />
+GFP.polyA with NgoMIV &amp; PstI-HF (2nd) gel purification</p>
+<p>&nbsp;</p>
        </div>
      </div>
      <div>
        <a href='#' class='head'>Week 4</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>26/8/2013</strong><br />
+  Sanger sequencing for P<i>cmv</i>.pSB1C3 (#1), MLS(RFC10).pSB1C3 (#4.3), and MLS(RFC25).pSB1C3 #1.1)<br />
+  Gel check for GFP.polyA (NgoMIV &amp; PstI-HF) for 1st &amp; 2nd<br />
+  GFP.polyA digestion with XbaI &amp; PstI-HF, run gel and gel purification<br />
+  P<i>cmv</i> digestion with SpeI &amp; PstI-HF, dephosphorylated<br />
+  MLS(RFC25).pSB1C3 digestion with AgeI &amp; PstI-HF, dephosphorylated<br />
+  Repeat Sequencing for MLS(RFC25).pSB1C3</p>
+<p>&nbsp;</p>
+<p><strong>27/8/2013</strong><br />
+  MLS(RFC25).pSB1C3 Sequencing (Cont.)<br />
+  Repeat Sequencing for P<i>cmv</i>.pSB1C3<br />
+  MLS(RFC10).pSB1C3 Sequencing (Cont.), transformed new plate with correct colony (#4.3)<br />
+  GFP.polyA(XbaI &amp; PstI-HF) gel Purification(Cont.), and gel check<br />
+  Gel check for P<i>cmv</i> (SpeI &amp; PstI-HF) &amp; MLS(RFC25).pSB1C3(AgeI &amp; PstI-HF)  <br />
+GFP(RFC25) digestion with EcoRI-HF &amp; PstI-HF, transformed new plate for correct colony (#2)</p>
+<p>&nbsp;</p>
+<p><strong>28/8/2013</strong><br />
+  Ligation of P<i>cmv</i> (S&amp;P) and GFP (X&amp;P), transformed<br />
+  Ligation of MLS (A&amp;P) and GFP (N&amp;P), transformed<br />
+  P<i>cmv</i>.pSB1C3 and MLS(RFC25) sequence confirmed<br />
+  GFP(RFC25) gel purification, EtOH precipitation<br />
+GFP(RFC25) nanodrop: concentration is too low, abandone</p>
+<p>&nbsp;</p>
+<p><strong>29/8/2013</strong><br />
+  Inoculation of MLS.GFP.polyA and CMV.GFP.polyA</p>
+<p>&nbsp;</p>
+<p><strong>30/8/2013</strong><br />
+  Plasmid extraction of MLS.GFP.polyA &amp; CMV.GFP.polyA<br />
+  Digestion check of MLS.GFP.polyA with EcoRV-HF &amp; NdeI, and CMV.GFP.polyA with EcoRV-HF &amp; AgeI-HF<br />
+Transfection of CMV.GFP.polyA #1</p>
+<p>&nbsp;</p>
        </div>
      </div>
@@ Line 498: / Line 726: @@
      <div>
        <a href='#' class='head'>Week 1</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>2/9/2013</strong><br />
+  Repeat Digestion check for MLS.GFP.polyA &amp; CMV.GFP.polyA<br />
+  Gel check of CMV.GFP.polyA: long gel, delayed since not enough time to run gel<br />
+  Digestion of MLS.GFP.polyA with XbaI &amp; PstI-HF</p>
+<p>&nbsp;</p>
+<p><strong>3/9/2013</strong><br />
+  Digestion check of CMV.GFP.polyA with EcoRV-HF &amp; AgeI-HF<br />
+  Run gel and gel purification of MLS.GFP.polyA (X &amp; P)<br />
+  P<i>cmv</i> digestion with SpeI and PstI-HF, dephosphorylated and PCR clean up<br />
+BBa_J176171 digestion with SpeI &amp; PstI-HF, dephosphorylation and PCR clean up</p>
+<p>&nbsp;</p>
+<p><strong>4/9/2013</strong><br />
+  GEl purification for  MLS.GFP.polyA</p>
+<p>&nbsp;</p>
+<p><strong>5/9/2013</strong><br />
+  Gel check for CMV.GFP.polyA (E&amp;P), CMV.pSB1C3 (E&amp;P), and GFP.polyA.pSB1C3(E&amp;P)<br />
+  Run gel for MLS.GFP.polyA(XbaI&amp;PstI-HF), CMV(SpeI&amp;PstI-HF), BBa_J176171(SpeI&amp;PstI-HF), and CMV.GFP.polyA (EcoRV-HF, AgeI-HF)</p>
+<p>&nbsp;</p>
+<p><strong>6/9/2013</strong><br />
+  Digestion of CMV.pSB1C3 (1) &amp; GFP.polyA(4) by EcoRI-HF &amp; PstI-HF<br />
+Gel check: decided that cmv is defect, will order new primer</p>
+<p>&nbsp;</p>
        </div>
      </div>
      <div>
        <a href='#' class='head'>Week 2</a>
-       <div class='content' align="left">Content 1
+       <div class='content' align="left">
+<p>&nbsp;</p>
+<p><strong>9/9/2013</strong><br />
+  pSB1C3 digestion with EcoRI-HF &amp; PstI-HF<br />
+  Inoculation of pEGFP-n1 (from confirmed colony spread plate)</p>
+<p>&nbsp;</p>
+<p><strong>10/9/2013</strong><br />
+  Gel purification of pSB1C3<br />
+Plasmid extraction of pEGFP-N1</p>
+<p>&nbsp;</p>
+<p><strong>12/9/2013</strong><br />
+  Digestion of MLS.GFP.polyA and GFP.polyA with SpeI &amp; PstI, dephosphorylated<br />
+  Amplify CMV with PCR using new primer</p>
+<p>&nbsp;</p>
        </div>
      </div>

Team:Hong Kong HKUST/notebook/mod3

From 2013.igem.org

Latest revision as of 23:14, 27 September 2013

Protein Trafficking Module's Notebook

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