Team:Queens/Notebook

From 2009.igem.org

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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 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;">16/06/2009</p>
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E;">16/06/2009</p>
<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. Please click on the Lab Journal you wish to view and either open in your browser or download to your computer.
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-Finished the sequence for the surface expression construct
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</pre></p>
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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 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/>
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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/>
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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/>
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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
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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/>
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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>
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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/>
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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/>
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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/>
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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/>
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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/>
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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/>
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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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<a name="Lab2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Laboratory Two: Kate, Mike</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="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 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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Last Updated: October 11, 2009 by Fr<sub>3</sub>P</center></font>
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Revision as of 23:36, 11 October 2009




16/06/2009

Please note that Lab Journal's are in PDF Format. Please click on the Lab Journal you wish to view and either open in your browser or download to your computer.


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Laboratory One: Harry, Bogdan, James, Bryant

Laboratory Two: Kate, Mike





Last Updated: October 11, 2009 by Fr3P

Retrieved from "http://2009.igem.org/Team:Queens/Notebook"