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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<td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Menu<i>
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Please note that Lab Journal's are in PDF Format.
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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="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>
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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="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>
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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, Chris</p></a>
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</tr>
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<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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<br/>
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E;font-weight:bold">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">
 
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-Lab Meeting
 
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-Finished the sequence for the surface expression construct
 
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-Finalized to-do list
 
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-Mini-prepped the following:
 
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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 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 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 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
 
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</pre>
 
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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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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>
 
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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
 
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</pre>
 
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</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">
 
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-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>
 
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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>
 
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</pre>
 
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</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>
 
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</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>
 
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</pre>
 
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</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">
 
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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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-Picked colonies from plates and made glycerol stocks
 
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</pre>
 
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</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 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
 
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and then run the gel again.
 
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</pre>
 
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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 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>
 
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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>
 
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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">
 
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-Picked colonies from plates and made broth culture which was left to grow overnight
 
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</pre>
 
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</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">
 
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-Purified the plasmids of parts 18-25 using Qiagen Spin MiniPrep Kit.
 
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</pre>
 
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</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">
 
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-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
 
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III. R0062 120μg/μL loaded 12μL
 
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IV. pSB1AT3 120μg/μL loaded 12μL
 
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V. J23119 120μg/μL loaded 12μL
 
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VI. J37034 120μg/μL loaded 12μL
 
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VII. K091204 120μg/μL loaded 12μL
 
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VIII. E0840 120μg/μL loaded 10μL
 
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IX. B0034 50μg/μL loaded 10μL
 
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X. F1610 50μg/μL loaded 10μL
 
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XI. I13522 120μg/μL loaded 10μL
 
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-Calculated the relative concentrations of the parts by comparing the bands with
 
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the ladder (The 5000bp ladder is about 120ng/μL)
 
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-Parts digested:
 
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I. R0062 Plux prefix EcoR1+Spe1
 
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II. pSB1AT3 backbone EcoR1+Pst1
 
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III. J23119 Pconst prefix EcoR1+Spe1
 
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IV. B0034 RBS suffix Xba1+Pst1
 
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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:
 
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-600ng of DNA
 
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-4μL restriction buffer
 
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-0.5μL EcoR1 and 0.5μL Spe1, or
 
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-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
 
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purified parts using QiaQuick PCR Purification Kit.
 
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-Stored the purified DNA in -20C overnight
 
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</pre>
 
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</p>
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">10/07/2009</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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-Ran a 1% Agarose gel electrophoresis to confirm the products of restriction
 
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digestions.
 
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-The expected bands of the parts did not show up. This is probably dude to
 
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the fact that most of the parts have lengths within 100bp and the QiaQuick
 
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PCR purification kit removes DNA below 100bp length.
 
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-The miniprep plasmids of the parts are digested again with corresponding
 
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restriction enzymes for 2 hours
 
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-Enzymes are deactivated (denatured) by heating at 65C
 
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</pre>
 
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</p>
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">13/07/2009</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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-Ligated the parts into the following constructs using the <i>T4 Ligase Protocol</i>
 
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-P<sub>const</sub> - RBS - LuxL - 2xSTOP
 
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-P<sub>lux</sub> - RBS
 
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-P<sub>const</sub> - RBS (Heme Oxygenase)
 
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-Terminator - RBS
 
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-Made broth culture from the VLA cDNA glycerol stock
 
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</pre>
 
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</p>
 
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<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">14/07/2009</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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-Purified the plasmid for ITGA4 using QiaMiniPrep Spin Kit
 
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-Recultured ITGA4-containing cells in broth
 
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</pre>
 
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</p>
 
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<br/>
 
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Last Updated: May 11, 2009 by Fr<sub>3</sub>P</center></font>
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Last Updated: October 20, 2009 by Fr<sub>3</sub>P</center></font>
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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