Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk"

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</table>
 
</table>
 
</li>
 
</li>
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<li> PCR program (using benchling annealing temperatures)
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<table style="width: 100%">
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<tr>
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<td><b>Temperature (C)</b></td>
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<td><b>Time (Min:sec)</b></td>
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</tr>
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<tr>
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<td><b>98</b></td>
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<td>0:30</td>
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</tr>
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<tr>
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<td><b>98 x 30 cycles</b></td>
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<td>0:10</td>
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</tr>
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<tr>
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<td><b>
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grad.
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55.4/58.4/61.4 C x 30
  
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</b></td>
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<td>0:20</td>
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</tr>
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<tr>
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<td><b>
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72 x 30
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</b></td>
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<td>0:30</td>
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</tr>
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<tr>
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<td><b>72(diluted to 1ng/uL)</b></td>
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<td>2:00</td>
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</tr>
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<tr>
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<td><b>12</b></td>
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<td>hold</td>
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</tr>
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</li>
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</li>
  
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<img width="300px" src="https://upload.wikimedia.org/wikipedia/commons/6/60/Gel_electrophoresis_2.jpg"/>
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<li>Had a slice of pizza</li>
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</ul>
 
</ul>
  
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</details>
 
</details>
  
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<details>
 
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<summary>May 3 - May 8</summary>
 
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<details>
 
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<summary>5/4 Transformation of Silk and Prom + Silk
 
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</summary>
 
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<ul>
 
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<li>Using Sri’s chemically competent cells
 
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</li>
 
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<li>Using ligation product from 4/30 (not purifying the ligation product as per Eric’s recommendation)
 
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</li>
 
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<li>Restriction digest</li>
 
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<img width="300px" src="https://upload.wikimedia.org/wikipedia/commons/6/60/Gel_electrophoresis_2.jpg"/>
 
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<li>Had a slice of pizza</li>
 
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</ul>
 
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</details>
 
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<details>
 
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<summary>Day 2</summary>
 
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<ul>
 
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<li>PCR</li>
 
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<li>Ran gel</li>
 
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<li>Restriction digest</li>
 
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</table>
 
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<ol>
 
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        <li>item 1</li>
 
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        <li>item 2</li>
 
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        <li>item 3</li>
 
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        <li>item 4</li>
 
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</ol>
 
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</ul>
 
  
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</details>
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<details>
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<summary>Day 3</summary>
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<ul>
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−
<li>PCR</li>
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−
<li>Ran gel</li>
+
−
<li>Restriction digest</li>
+
−
<li>Had a slice of pizza</li>
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−
</ul>
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−
</details>
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</details>
 
</details>
  

Revision as of 06:03, 14 May 2015

iGEM UCLA




Honeybee Silk Notebook

April 26 - May 2
4/26 PCR off honey bee gene block
  • See Benchling for the g block sequence and primer reference code https://benchling.com/s/p7bClpzQ/edit
  • The goal of this is to clone 2 different constructs and prepare them as biobricks, one that is just the silk gene, and the other that as the promoter, rbs, etc.. required for protein expression.
  • Using primers p3, p7, and p8. (See Benchling link)
  • PCR Reaction 1 (q5 polymerase kit): primers honeybee p#7 and honeybee p#8 with gBlock, and honeybee p#3 and honeybee p#8 amplify just the honeybee coding region and the prom + coding region respectively
    Component Volume
    5X Q5 Reaction Buffer 5
    10 mM dNTPs 0.5
    10 uM Forward (primer 3/7) 1.25
    10 uM Reverse (primer 8) 1.25
    Template (diluted to 1ng/uL) 0.5
    Q5 High Fidelity DNA Polymerase 0.25
    Nuclease Free Water 16.25
  • PCR program (using benchling annealing temperatures)


    Temperature (C) Time (Min:sec)
    98 0:30
    98 x 30 cycles 0:10
    grad. 55.4/58.4/61.4 C x 30 0:20
    72 x 30 0:30
    72(diluted to 1ng/uL) 2:00
    12 hold