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

Line 16: Line 16:
 
<li>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.
 
<li>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.
 
</li>
 
</li>
−
<li>Restriction digest</li>
+
<li>Using primers p3, p7, and p8. (See Benchling link)</li>
 +
<li>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
 +
<table style="width: 100%">
 +
<tr>
 +
<td><b>Component</b></td>
 +
<td><b>Volume</b></td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>5X Q5 Reaction Buffer</b></td>
 +
<td>5</td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>10 mM dNTPs</b></td>
 +
<td>0.5</td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>
 +
10 uM Forward (primer 3/7)
 +
</b></td>
 +
<td>1.25</td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>
 +
10 uM Reverse (primer 8)
 +
</b></td>
 +
<td>1.25</td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>Template (diluted to 1ng/uL)</b></td>
 +
<td>0.5</td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>
 +
Q5 High Fidelity DNA Polymerase
 +
</b></td>
 +
<td>0.25</td>
 +
 +
</tr>
 +
<tr>
 +
<td><b>Nuclease Free Water</b></td>
 +
<td>16.25</td>
 +
 +
</tr>
 +
</table>
 +
</li>
 +
 
 +
 
 
<img width="300px" src="https://upload.wikimedia.org/wikipedia/commons/6/60/Gel_electrophoresis_2.jpg"/>
 
<img width="300px" src="https://upload.wikimedia.org/wikipedia/commons/6/60/Gel_electrophoresis_2.jpg"/>
 
<li>Had a slice of pizza</li>
 
<li>Had a slice of pizza</li>

Revision as of 05:44, 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
  • Had a slice of pizza
May 3 - May 8
5/4 Transformation of Silk and Prom + Silk
  • Using Sri’s chemically competent cells
  • Using ligation product from 4/30 (not purifying the ligation product as per Eric’s recommendation)
  • Restriction digest
  • Had a slice of pizza
Day 2
  • PCR
  • Ran gel
  • Restriction digest
    • Item Item Item Item
    Item Item Item Item
    Item Item Item Item
    1. item 1
    2. item 2
    3. item 3
    4. item 4
Day 3
  • PCR
  • Ran gel
  • Restriction digest
  • Had a slice of pizza