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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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 </head>
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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>
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 </div>
 <br><br><br><br><br><br><br><br>
-<div id="title"><h3>Protein Trafficking Module's Notebook</h3></div>
+<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 349: / Line 372: @@
 <p>&nbsp;</p>
 <p><strong>24-6-2013</strong> <br />
-   Inoculation of pcmv/myc/mito </p>
+   Inoculation of pCMV/myc/mito </p>
 <p>&nbsp;</p>
 <p><strong>25-6-2013</strong> <br />
-   Plasmid extraction for pcmv/myc/mito <br />
+   Plasmid extraction for pCMV/myc/mito <br />
-   Digestion of pcmv plasmid extraction product with PstI-HF and NotI-HF <br />
+   Digestion of pCMV plasmid extraction product with PstI-HF and NotI-HF <br />
-   Gel electrophoresis of pcmv digested product <br />
+   Gel electrophoresis of pCMV digested product <br />
-Gel check for pcmv digestion product </p>
+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 purification of pCMV gel product <br />
-   Gel check of pcmv gel purified product <br />
+   Gel check of pCMV gel purified product <br />
-   PCR clone MLS from pcmv/myc/mito <br />
+   PCR clone MLS from pCMV/myc/mito <br />
 Transformation of pEGFP-N1 with SURE </p>
 <p>&nbsp;</p>
@@ Line 424: / Line 447: @@
 <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 />
+   Inoculation of MLS.pSB1C3 <br />
    Plasmid extraction of pEGFP-N1 <br />
    Digestion of pEGFP-N1 with NotI-HF &amp; PstI-HF <br />
@@ Line 436: / Line 459: @@
 <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 />
+   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>
+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 />
+   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 />
+   Plasmid extraction of pCMV/myc/mito.EGFP <br />
-pcmv/myc/mito.EGFP transformation check by digesting with PstI &amp; NotI</p>
+pCMV/myc/mito.EGFP transformation check by digesting with PstI &amp; NotI</p>
 <p>Spread plate for confirmed colony </p>
 <p>&nbsp;</p>
@@ Line 457: / Line 480: @@
 <p>&nbsp;</p>
 <p><strong>15/7/2013</strong><br />
-   pcmv/myc/mito.GFP inoculation</p>
+   pCMV/myc/mito.GFP inoculation</p>
 <p>&nbsp;</p>
 <p><strong>16/7/2013</strong><br />
-   Plasmid Extraction of pcmv/myc/mito.GFP<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>
+   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 />
+   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(25) PCR product, PCR clean up<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>
@@ Line 486: / Line 509: @@
    Gel check of pSB1C3: band is too pale, repeat inoculation<br />
    Inoculation of pSB1C3.J04450<br />
-   Repeat PCR of MLS(25)<br />
+   Repeat PCR of MLS(RFC25)<br />
    Gel check of PCR product and PCR clean up<br />
-   Digestion of MLS(25) with EcoRI &amp; SpeI, run gel</p>
+   Digestion of MLS(RFC25) with EcoRI &amp; SpeI, run gel</p>
 <p>&nbsp;</p>
 <p><strong>23-7-2013<br />
-   </strong>MLS(25) Gel purification (Cont), and gel check<br />
+   </strong>MLS(RFC25) Gel purification (Cont), and gel check<br />
    pSB1C3 plasmid extraction<br />
 Digestion of pSB1C3 with EcoRI &amp; SpeI</p>
@@ Line 497: / Line 520: @@
 <p><strong>24-7-2013</strong><br />
    pSB1C3 Gel purification (Cont.)<br />
-MLS(25) &amp; pSB1C3 ligation, and transform to CAM plate</p>
+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(25)<br />
+   Colony PCR of MLS.pSB1C3(RFC25)<br />
-Inoculation of MLS.pSB1c3(25)</p>
+Inoculation of MLS.pSB1C3(RFC25)</p>
 <p>&nbsp;</p>
 <p><strong>26-7-2013</strong><br />
-   Plasmid extraction of MLS.pSB1c3(25)<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(25) colony PCR product<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>
@@ Line 517: / Line 540: @@
 <p>&nbsp;</p>
 <p><strong>29-7-2013</strong><br />
-   Repeat digestion of pSB1C3.MLS(25) with EcoRI and SpeI, gel check and Sanger sequence<br />
+   Repeat digestion of pSB1C3.MLS(RFC25) with EcoRI and SpeI, gel check and Sanger sequence<br />
-   Spread plate of GFP(25) transformed colonies</p>
+   Spread plate of GFP(RFC25) transformed colonies</p>
 <p>&nbsp;</p>
 <p><strong>30-7-2013</strong><br />
-   pSB1C3.MLS (25) digestion with EcoRI and SpeI<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(25) colonies</p>
+Streak plate of EGFP(RFC25) colonies</p>
 <p>&nbsp;</p>
 <p><strong>31-7-2013</strong><br />
-   pSB1C3.MLS (25) Sanger sequence(Cont.)<br />
+   pSB1C3.MLS (RFC25) Sanger sequence(Cont.)<br />
-GFP (25) inoculation</p>
+GFP (RFC25) inoculation</p>
 <p>&nbsp;</p>
 <p><strong>01-08-2013</strong><br />
-   GFP(25) plasmid extraction<br />
+   GFP(RFC25) plasmid extraction<br />
    Prepare the glycerol stock and stored it<br />
-Digestion of GFP(25) with EcoRI and SpeI, gel electrophoresis</p>
+Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis</p>
 <p>&nbsp;</p>
 <p><strong>02-08-2013</strong><br />
-   GFP (25) plasmid extraction<br />
+   GFP (RFC25) plasmid extraction<br />
-   Digestion of GFP(25) with EcoRI and SpeI, gel electrophoresis<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>
@@ Line 552: / Line 575: @@
 <p>&nbsp;</p>
 <p><strong>5-8-2013</strong><br />
-   GFP (25) PCR Check with VR VF2, gel check (over ran)<br />
+   GFP (RFC25) PCR Check with VR VF2, gel check (over ran)<br />
    BBa_J176171 digestion using SpeI and PstI-HF to extract the backbone<br />
    PolyA / BBa_K404108 digestion using XbaI and PstI-HF<br />
@@ Line 558: / Line 581: @@
 <p>&nbsp;</p>
 <p><strong>6-8-2013</strong><br />
-   Repeat PCR check of GFP (25)<br />
+   Repeat PCR check of GFP (RFC25)<br />
    pSB1C3 plasmid extraction<br />
    PolyA / BBa_K404108 gel purification and gel check<br />
    BBa_J176171 PCR clean up<br />
    <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 />
-PCR amplify MLS in RFC 10 and 25 format</p>
+PCR amplify MLS in RFC 10 and RFC25 format</p>
 <p>&nbsp;</p>
 <p><strong>7-8-2013</strong><br />
-   MLS (RFC 10 &amp; 25) PCR clean up<br />
+   MLS (RFC 10 &amp; RFC25) PCR clean up<br />
    Nanodrop and gel check for the PCR clean up product<br />
    Digestion using EcoRI and PstI<br />
    PolyA / BBa_K404108 gel check<br />
    pSB1C3 digestion with EcoRI &amp; PstI<br />
-GFP(25) digestion with EcoRI &amp; SpeI</p>
+GFP(RFC25) digestion with EcoRI &amp; SpeI</p>
 <p>&nbsp;</p>
 <p><strong>8/8/2013</strong><br />
-   Gel purification of<strong> </strong>GFP25 &amp; pSB1C3, then nanodrop and gel check <br />
+   Gel purification of<strong> </strong>GFP(RFC25) &amp; pSB1C3, then nanodrop and gel check <br />
    polyA digestion with EcoRI and XbaI, and PCR clean up<br />
 Inoculation of BBa_K929000 &amp; BBa_J52034</p>
@@ Line 594: / Line 617: @@
 <p>&nbsp;</p>
 <p><strong>13/08/2013</strong><br />
-   MLS10.psb1c3 &amp; gpf.polyA plasmid extraction<br />
+   MLS(RFC10).pSB1C3 &amp; gpf.polyA plasmid extraction<br />
-   Digestion of MLS10.psb1c3 &amp; gpf.polyA with EcoRI-HF and PstI-HF <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 />
-   MLS25.pSB1C3<br />
+   MLS(RFC25).pSB1C3<br />
-Streak plate with transformed MLS25.pSB1C3 to purify colony</p>
+Streak plate with transformed MLS(RFC25).pSB1C3 to purify colony</p>
 <p>&nbsp;</p>
 <p><strong>14/08/2013</strong><br />
-   Inoculation of MLS25.pSB1C3 &amp; GFP.PolyA</p>
+   Inoculation of MLS(RFC25).pSB1C3 &amp; GFP.PolyA</p>
 <p>&nbsp;</p>
 <p><strong>15/08/2013</strong><br />
    pSB1C3 gel purification<br />
-   MLS10.pSB1C3 Sanger sequencing (did not submit as the colony turn out to be not single)<br />
+   MLS(RFC10).pSB1C3 Sanger sequencing (did not submit as the colony turn out to be not single)<br />
-   Plasmid extraction of MLS25.pSB1C3 &amp; GFP<br />
+   Plasmid extraction of MLS(RFC25).pSB1C3 &amp; GFP<br />
-Digestion check of MLS25 with EcoRI &amp; PstI; GFP.polya with ApaLI &amp; AgeI HF</p>
+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 />
+   pCMV (Amplify by PCR from pEGFP-N1) PCR clean up<br />
    pSB1C3 dephosphorylation<br />
-   Run gel of MLS25.psb1c3 &amp; gpf.polya<br />
+   Run gel of MLS(RFC25).pSB1C3 &amp; gpf.polya<br />
-   MLS25.psb1c3 PCR check<br />
+   MLS(RFC25).pSB1C3 PCR check<br />
-   Streak plate with transformed MLS10.pSB1C3 to get single colony <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 />
+   Inoculation of pEGFP-N1, pCMV/my/mito.GFP, pCMV/myc/mito<br />
 Streak plate with transformed pEGFP-N1</p>
 <p>&nbsp;</p>
@@ Line 628: / Line 651: @@
    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 />
+   Streak pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP to AMP plate<br />
-   MLS25.pSB1C3 &amp; GFP.polya digestion with AgeI &amp; ApaLI, and transformation of the correct colony<br />
+   MLS(RFC25).pSB1C3 &amp; GFP.polya digestion with AgeI &amp; ApaLI, and transformation of the correct colony<br />
-   MLS25.pSB1C3 Sanger Sequencing</p>
+   MLS(RFC25).pSB1C3 Sanger Sequencing</p>
 <p>&nbsp;</p>
 <p><strong>20/8/2013</strong><br />
-   MLS25.pSB1C3 Sanger Sequencing(Cont.)<br />
+   MLS(RFC25).pSB1C3 Sanger Sequencing(Cont.)<br />
-   Plasmid extraction of pEGFP-N1, pcmv/myc/mito.GFP and pcmv/myc/mito.EGFP<br />
+   Plasmid extraction of pEGFP-N1, pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP<br />
-   MLS25.psb1C3 digestion with AgeI-HF &amp; PstI-HF<br />
+   MLS(RFC25).pSB1C3 digestion with AgeI-HF &amp; PstI-HF<br />
-   GFP.polya.psb1c3(1st try) digestion with NgoMIV &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>
@@ Line 642: / Line 665: @@
    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 />
-   pCMV.psb1c3(2nd) ligation, and transformation to DH5alpha<br />
+   P<i>cmv</i>.pSB1C3(2nd) ligation, and transformation to DH5alpha<br />
-MLS10 bacteria inoculation</p>
+MLS(RFC10) bacteria inoculation</p>
 <p>&nbsp;</p>
 <p><strong>22/08/2013</strong><br />
-   MLS10.pSB1c3 plasmid extraction, PCR check with VR VF2<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 />
-   pCMV.pSB1C3 (3rd) ligation and transformation<br />
+   P<i>cmv</i>.pSB1C3 (3rd) ligation and transformation<br />
-   Inoculation of pCMV.pSB1C3 (2nd)<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 MLS10.pSB1C3 and MLS25.pSB1C3<br />
+   Sanger Sequencing for MLS(RFC10).pSB1C3 and MLS(RFC25).pSB1C3<br />
-   pCMV.pSB1C3 (2nd) plasmid extraction, and digestion check with EcoRI &amp; NdeI<br />
+   P<i>cmv</i>.pSB1C3 (2nd) plasmid extraction, and digestion check with EcoRI &amp; NdeI<br />
-   Streak the right colony of pCMV.pSB1C3 (2nd) into plate <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>
@@ Line 666: / Line 689: @@
 <p>&nbsp;</p>
 <p><strong>26/8/2013</strong><br />
-   Sanger sequencing for pCMV.pSB1C3 (#1), MLS10.pSB1C3 (#4.3), and MLS25.pSB1C3 #1.1)<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 />
-   pCMV digestion with SpeI &amp; PstI-HF, dephosphorylated<br />
+   P<i>cmv</i> digestion with SpeI &amp; PstI-HF, dephosphorylated<br />
-   MLS25.pSB1C3 digestion with AgeI &amp; PstI-HF, dephosphorylated<br />
+   MLS(RFC25).pSB1C3 digestion with AgeI &amp; PstI-HF, dephosphorylated<br />
-   Repeat Sequencing for MLS25.pSB1C3</p>
+   Repeat Sequencing for MLS(RFC25).pSB1C3</p>
 <p>&nbsp;</p>
 <p><strong>27/8/2013</strong><br />
-   MLS25.pSB1C3 Sequencing (Cont.)<br />
+   MLS(RFC25).pSB1C3 Sequencing (Cont.)<br />
-   Repeat Sequencing for pCMV.pSB1C3<br />
+   Repeat Sequencing for P<i>cmv</i>.pSB1C3<br />
-   MLS10.pSB1C3 Sequencing (Cont.), transformed new plate with correct colony (#4.3)<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 pCMV (SpeI &amp; PstI-HF) &amp; MLS25.psb1c3(AgeI &amp; PstI-HF)  <br />
+   Gel check for P<i>cmv</i> (SpeI &amp; PstI-HF) &amp; MLS(RFC25).pSB1C3(AgeI &amp; PstI-HF)  <br />
-GFP25 digestion with EcoRI-HF &amp; PstI-HF, transformed new plate for correct colony (#2)</p>
+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 pCMV (S&amp;P) and GFP (X&amp;P), transformed<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 />
-   pCMV.pSB1C3 and MLS(25) sequence confirmed<br />
+   P<i>cmv</i>.pSB1C3 and MLS(RFC25) sequence confirmed<br />
-   GFP25 gel purification, EtOH precipitation<br />
+   GFP(RFC25) gel purification, EtOH precipitation<br />
-GFP25 nanodrop: concentration is too low, abandone</p>
+GFP(RFC25) nanodrop: concentration is too low, abandone</p>
 <p>&nbsp;</p>
 <p><strong>29/8/2013</strong><br />
@@ Line 713: / Line 736: @@
    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 />
-   pCMV digestion with SpeI and PstI-HF, dephosphorylated and PCR clean up<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>
@@ Line 724: / Line 747: @@
 <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 />
+   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>
@@ Line 738: / Line 761: @@
 <p>&nbsp;</p>
 <p><strong>10/9/2013</strong><br />
-   Gel purification of pSB1c3<br />
+   Gel purification of pSB1C3<br />
 Plasmid extraction of pEGFP-N1</p>
 <p>&nbsp;</p>

Team:Hong Kong HKUST/notebook/mod3

From 2013.igem.org

Latest revision as of 23:14, 27 September 2013

Protein Trafficking Module's Notebook

June 2013

July 2013

August 2013

September 2013