Team:Queens/Notebook

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<table style="background-color:#922334; position:relative; overflow:auto; left:200px; top:-215px; width:750px">
<table style="background-color:#922334; position:relative; overflow:auto; left:200px; top:-215px; width:750px">
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<tr>
 
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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Menu<i>
 
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</p></td>
 
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</tr>
 
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<tr>
 
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<td align="left">
 
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<a href="#Lab1"><p style="font-size:120%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a>
 
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<a href="#Lab2"><p style="font-size:120%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a>
 
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<a href="#Tech"><p style="font-size:120%;font-family:corbel;color:#ECB528;font-weight:bold">Tech Support and Laboratory Three: Parthiv, Chris P., Chris Y.</p></a>
 
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</td>
 
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</tr>
 
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<tr>
 
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<td>
 
<br/>
<br/>
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<br/>
 
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</td>
 
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</tr>
 
<tr>
<tr>
<td align="left">
<td align="left">
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<a name="Lab1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E;">
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<br/>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E;">16/06/2009</p>
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Lab Meeting
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Please note that Lab Journal's are in PDF Format.  
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-Finished the sequence for the surface expression construct
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Please click on the Lab Journal you wish to view.
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-Finalized to-do list
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</pre></p></td>
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-Mini-prepped the following:
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</tr>
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-Linker K157013 <i>Plate 3, Well 3G, plasmid Bba_K157000(A)</i>
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-TEV Protease
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-I712078 (C-Terminus) <i>Plate 2, Well 14M, plasmid J70003 (A)</i>
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-I712077 (N-Terminus) <i>Plate 2, Well 14K, plasmid J70003 (A)</i>
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-pLux
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-R0062 (not leaky) <i>Plate 1, Well 6O, plasmid pSB1A2 (A)</i>
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-R1062 (median strength in the absence of luxR/HSL)
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<i>Plate 1, Well 8G, plasmid pSB1A2 (A)</i>
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-HO-pcyA
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-K098010 <i>Plate 3, Well 11N, plasmid pSB3k3 (K)</i>
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-Terminator
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-B0015 <i>Plate 1, Well 23L, plasmid pSB1AK3 (AK)</i>
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-RFP
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-E1010 <i>Plate 1, Well 18F, plasmid pSB2K3 (K)</i>
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</pre>
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</p>
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<p/>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">17/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Transformed the following parts into Top10
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1. K157013 <i>Plate 3, Well 3G, K157000, Res:A</i>
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2. I712078 <i>Plate 2, well 14M, J70003, Res:A</i>
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3. I712077 <i>Plate 2, Well 14K, J70003, Res:A</i>
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4. R0062 <i>Plate 1, Well 6O, pSB1A2, Res:A</i>
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5. R1062 <i>Plate 1, Well 8G, pSB1A2, Res:A</i>
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6. K098910 <i>Plate 3, Well 11N, pSB3K3, Res:A</i>
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7. B0015 <i>Plate 1, Well 23L, pSB1AK3, Res:AK</i>
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8. E1010 <i>Plate 2, Well 18F, pSB2K3, Res:K</i>
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-Left in 37C for four hours and left on lab bench at room temperature overnight
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</pre>
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</p>
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<p/>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">18/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-No colonies were observered on the plates
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-Left the plates in 37C for four hours
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-Picked one colony from each plate and made overnight broth culture for glycerol stock.
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-K098010 plate did not grow, so it was left in 37C overnight.
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</pre>
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</p>
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<p/>
+
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">19/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
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-Made the glycerol stocks from the overnight broth cultures
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-Started broth culture for K098010
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-Re-transformed the E1010 in Top10
+
-
</pre>
+
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</p>
+
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<p/>
+
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">22/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
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-Restarted the broth culture for K098010 because it was overgrown (already in stationary phase)
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-Observed no colonies on E1010 plate. Discovered that E1010 should be on Kanamycin resistance plate.
+
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+
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Things to Do:
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-Get out the standard plasmid backbones
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-High copy number assembly plasmid backbone
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-pSB1A3 <i>Plate 1, Well 1K</i>
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</pre>
+
-
</p>
+
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<p/>
+
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">23/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Submitted VLA construct to Mr. GENE for synthesis
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-Placed orders for PCR primers
+
-
</pre>
+
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</p>
+
-
<p/>
+
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">24/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Transformed the following parts into Top10
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9.  RBS-LuxR J37033 <i>Plate 3, Well 4O, pSB1A2, Res:A</i>
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-
10. RBS-LuxL-ter F1610 <i>Plate 2, Well 24G, pSB1AK3, Res:AK</i>
+
-
11. RBS-LuxL K081008 <i>Plate 2, Well 10L, pSB1A2, Res:A</i>
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12. RBS-LuxR-ter I0462 <i>Plate 1, Well 8O, pSB1A2, Res:A</i>
+
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13. Pconst J23119 <i>Plate 1, Well 18A, pSB1A2, Res:A</i>
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-
14. LuxR C0062 <i>Plate 1, Well 4O, pSB1A2, Res:A</i>
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">25/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
-Purchased QiaMiniPrep Kit from BioBar
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-
-Picked colonies from plates and re-cultured in broth; left overnight
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-
-Ordered ITGA4 cDNA plasmid from OpenBioSystem
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-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">26/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
-Made glycerol stocks for parts transformed 23/06/2009
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-Designed primer for PCR out VLA cDNA plasmid and primer for pLux-RBS-HindIII
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-Transformed the following parts into Top10 and left the plates in 37C overnight.
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15. RBS B0034 <i>Plate 1, Well 2M, pSB1A2, Res:A</i>
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16. HemeC I716154 <i>Plate 1, Well 17B, pSB1A2, Res:A</i>
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-
17. HemeD I716155 <i>Plate 1, Well 17D, pSB1A2, Res:A</i>
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
+
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">27/06/2009</p>
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-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Picked colonies from plates and made glycerol stocks
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">28/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Transformed gycerol stocks into broth culture
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</pre>
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</p>
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<p/>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">29/06/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Purified plasmids containing parts 1-17 from Top10 using Qiagen Spin MiniPrep Kit.
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-Performed Agarose Gel Electrophoresis to check the size of the parts. Bends did
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not migrate very far, possible due tot he fact that circular plasmids migrate very
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slowly. We decided to digest the plasmids with a BioBrick restriction enzyme
+
-
and then run the gel again.
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-
</pre>
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</p>
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<p/>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">02/07/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Re-ran the Agarose Gel Electrophoresis to ckeck the size of the parts. It worked.
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-
</pre>
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</p>
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<p/>
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">06/07/2009</p>
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<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
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-Transformed the following parts into Top10
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18. GFP E0040 <i>Plate 1, Well 14K, pSB1A2, Res:A</i>
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19. GFP constr. E0840 <i>Plate 1, Well 12O, pSB1A2, Res:A</i>
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-
20. pTet+GFP I13522 <i>Plate 2, Well 8A, pSB1A2, Res:A</i>
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21. LuxR constr. K091204 <i>Plate 2, Well 8J, pSB1A2, Res:A</i>
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22. LuxL+GFP J37034 <i>Plate 2, Well 7I, pSB1A2, Res:AK</i>
+
-
23. pSB1AC3 <i>Plate 1, Well 11A Res:AC</i>
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24. pSB1AK3 <i>Plate 1, Well 13A Res:AK</i>
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25. pSB1AT3 <i>Plate 1, Well 15A, Res:AT</i>
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-
</pre>
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-
</p>
+
-
<p/>
+
-
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">07/07/2009</p>
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-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Picked colonies from plates and made broth culture which was left to grow overnight
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">08/07/2009</p>
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-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Purified the plasmids of parts 18-25 using Qiagen Spin MiniPrep Kit.
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">09/07/2009</p>
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-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
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-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Ran a 1% Agarose gel electrophoresis to confirm the plasmid lengths of parts 18 to 25
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-Gel loading and concentrations:
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I. E0040 120μg/μL loaded 12μL
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-
II. pSB1AC3 120μg/μL loaded 12μL
+
-
III. R0062 120μg/μL loaded 12μL
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-
IV. pSB1AT3 120μg/μL loaded 12μL
+
-
V. J23119 120μg/μL loaded 12μL
+
-
VI. J37034 120μg/μL loaded 12μL
+
-
VII. K091204 120μg/μL loaded 12μL
+
-
VIII. E0840 120μg/μL loaded 10μL
+
-
IX. B0034 50μg/μL loaded 10μL
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-
X. F1610 50μg/μL loaded 10μL
+
-
XI. I13522 120μg/μL loaded 10μL
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-
-Calculated the relative concentrations of the parts by comparing the bands with
+
-
the ladder (The 5000bp ladder is about 120ng/μL)
+
-
-Parts digested:
+
-
I. R0062 Plux prefix EcoR1+Spe1
+
-
II. pSB1AT3 backbone EcoR1+Pst1
+
-
III. J23119 Pconst prefix EcoR1+Spe1
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-
IV. B0034 RBS suffix Xba1+Pst1
+
-
V. F1610 RBS-LuxI-STOP suffix Xba1+Pst1
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-
VI. B0015 Terminator prefix EcoR1+Spe1
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-
-Restriction digestion mix recipe:
+
-
-600ng of DNA
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-
-4μL restriction buffer
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-
-0.5μL EcoR1 and 0.5μL Spe1, or
+
-
-0.5μL Xba1 and 0.5μL Pst1
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-
-Up to 35μL of ddH<sub>2</sub>0
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-
-Digested the parts using corresponding BioBrick restriction enzymes, and then
+
-
purified parts using QiaQuick PCR Purification Kit.
+
-
-Stored the purified DNA in -20C overnight
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
+
-
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">10/07/2009</p>
+
-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Ran a 1% Agarose gel electrophoresis to confirm the products of restriction
+
-
digestions.
+
-
-The expected bands of the parts did not show up. This is probably dude to
+
-
the fact that most of the parts have lengths within 100bp and the QiaQuick
+
-
PCR purification kit removes DNA below 100bp length.
+
-
-The miniprep plasmids of the parts are digested again with corresponding
+
-
restriction enzymes for 2 hours
+
-
-Enzymes are deactivated (denatured) by heating at 65C
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
+
-
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">13/07/2009</p>
+
-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Ligated the parts into the following constructs using the <i>T4 Ligase Protocol</i>
+
-
-P<sub>const</sub> - RBS - LuxL - 2xSTOP
+
-
-P<sub>lux</sub> - RBS
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-
-P<sub>const</sub> - RBS (Heme Oxygenase)
+
-
-Terminator - RBS
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-
-Made broth culture from the VLA cDNA glycerol stock
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
+
-
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">14/07/2009</p>
+
-
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
+
-
-Purified the plasmid for ITGA4 using QiaMiniPrep Spin Kit
+
-
-Recultured ITGA4-containing cells in broth
+
-
</pre>
+
-
</p>
+
-
<p/>
+
-
 
+
<br/>
<br/>
-
</td>
 
-
</tr>
 
<tr>
<tr>
<td align="left">
<td align="left">
-
<a name="Lab2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Laboratory Two: Kate, Mike</p></a>
+
<a href="https://static.igem.org/mediawiki/2009/5/5d/QueensLabNotesHarryBryantJamesBogdan.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a>
-
<br/>
+
<a href="https://static.igem.org/mediawiki/2009/0/0e/QueensLabNotesKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a>
-
 
+
-
 
+
-
 
+
-
</br>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td align="left">
+
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<a name="Tech"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Tech Support and Laboratory Three: Parthiv, Chris P., Chris Y.</p></a>
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Latest revision as of 02:03, 21 October 2009





Please note that Lab Journal's are in PDF Format. 
Please click on the Lab Journal you wish to view.

Laboratory One: Harry, Bogdan, James, Bryant

Laboratory Two: Kate, Mike






Last Updated: October 20, 2009 by Fr3P