Team:Caltech/Week 2

From 2011.igem.org

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<p>Lab walkthrough<br/>
<p>Lab walkthrough<br/>
Setting up lab, stock supplies, preparation of LB-amp plates<br/>
Setting up lab, stock supplies, preparation of LB-amp plates<br/>
-
Test extraction of DNA from BioBrick wells into cloning plates<br/>
+
Test extraction of DNA from BioBrick wells into cloning plates ([http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_K123001 K123001], [http://partsregistry.org/Part:BBa_K123002 K123002], [http://partsregistry.org/Part:BBa_K123003 K123003])<br/>
Initial enrichment cultures of collected samples on minimal media with BPA or 17a-ethynylestradiol</p>
Initial enrichment cultures of collected samples on minimal media with BPA or 17a-ethynylestradiol</p>
== June 21 ==
== June 21 ==
-
<p>MoBio kit extraction of DNA from collected samples<br/>
+
<p>MoBio Powermax Soil kit extraction of DNA from collected samples<br/>
Initial enrichment cultures of samples in minimal media with nonylphenol or DDT<br/>
Initial enrichment cultures of samples in minimal media with nonylphenol or DDT<br/>
Geneious and primer design tutorial</p>
Geneious and primer design tutorial</p>
-
 
+
===Results===
 +
MoBio Extraction
 +
<table border="1">
 +
<tr>
 +
<th>Sample</th>
 +
<th>Concentration (ng/µl)</th>
 +
</tr>
 +
<tr>
 +
<td><center>1</center></td>
 +
<td><center>4.5</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>2</center></td>
 +
<td><center>4.1</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>4</center></td>
 +
<td><center>0.0</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>5</center></td>
 +
<td><center>0.7</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>6</center></td>
 +
<td><center>13.7</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>7</center></td>
 +
<td><center>5.8</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>9</center></td>
 +
<td><center>58.7</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>10</center></td>
 +
<td><center>11.4</center></td>
 +
</tr>
 +
</table>
 +
Locations 3 and 8 were omitted from the MoBio Powermax Soil Extraction procedure because they were liquid samples.
== June 22 ==
== June 22 ==
-
<p>Miniprep and sequencing of selected BioBricks ([http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_K123001 K123001], [http://partsregistry.org/Part:BBa_K123002 K123002], [http://partsregistry.org/Part:BBa_K123003 K123003]) <br/>
+
<p>Miniprep and sequencing of selected BioBricks (BBa_K123000, BBa_K123001, BBa_K123002, BBa_K123003)<br/>
Preparation of competent cells</p>  
Preparation of competent cells</p>  
 +
Results of OD measurements:
 +
<table border="1">
 +
<tr>
 +
<th> Part </th>
 +
<th>Concentration (ng/µl)</th>
 +
</tr>
 +
<tr>
 +
<td><center>K123000</center></td>
 +
<td><center>326.6</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>K123001</center></td>
 +
<td><center>293.8</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>K123002</center></td>
 +
<td><center>152.3</center></td>
 +
</tr>
 +
<tr>
 +
<td><center>K123003</center></td>
 +
<td><center>402.6</center></td>
 +
</tr>
 +
</table>
==June 23==
==June 23==
-
<p>Transform T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]]), and double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) biobricks for test sequences for BisA and BisB degradation genes ([http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001])<br/>
+
<p>Transformation of T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]]), and double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) biobricks for test sequences for BisA and BisB degradation genes ([http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001])<br/>
-
Retest competent cells made June 22nd with [http://partsregistry.org/Part:BBa_K123001 K123001] and pUC<br/>
+
Retested competent cells made June 22nd with [http://partsregistry.org/Part:BBa_K123001 K123001] and pUC<br/>
-
Transfer 0.5 mL aliquots of BPA and 17a-ethynylestradiol cultures from June 20 to new tubes of minimal media<br/>
+
Transfered 0.5 mL aliquots of BPA and 5mL 17a-ethynylestradiol cultures from June 20 to new tubes of minimal media<br/>
-
Design primers for test sequence of BisdA (pNT001)<br/>
+
Designed primers for test sequence of BisdA (pNT001)<br/>
-
Design primers to continue sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003])<br/>
+
Designed primers to continue sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003])<br/><br/>
We determined that sequences for [http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001] have correct protein coding but different codons than in the sequences shown online </p>
We determined that sequences for [http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001] have correct protein coding but different codons than in the sequences shown online </p>
==June 24==
==June 24==
-
<p>Transform Tet repressible promoter [http://partsregistry.org/Part:BBa_R0040 R0040]<br/>
+
<p>Transformation of Tet repressible promoter [http://partsregistry.org/Part:BBa_R0040 R0040]<br/>
-
Make plain LB and pour plain LB plates<br/>
+
Preparation of LB, LB (no antibiotic) plates, and SOC media<br/>
-
Make SOC media<br/>
+
Transfer of 5 mL DDT and nonylphenol cultures from June 21 to new tubes of minimal media <br/>
-
Transfer 0.5 mL DDT and nonylphenol cultures from June 21 to new tubes of minimal media <br/>
+
Design of reverse primer for continued sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003]) <br/>
-
Wash dishes from last year <br/>
+
Update Wiki with protocols we have been using<br/><br/>
-
Design reverse primer for continued sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003]) <br/>
+
We determined that our competent cells are functional have a cfu/ug DNA of 6.6 x 10^6.</p>  
-
Update Wiki with protocols we have been using</p>  
+
==June 25==
==June 25==
-
<p>Take out plates from June 24 </p>
+
<p>Take out plates from June 24</p>
-
 
+
-
==June 26==
+
-
<p> Start overnight cultures of T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]), double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) and Tet Promoter ([http://partsregistry.org/Part:BBa_R0040 R0040])</p>
+
}}
}}

Latest revision as of 21:56, 1 August 2011


Caltech iGEM 2011



Home

Project

Data

Parts

Team

Notebook

Biosafety

Human Impact

References

Support

June 19

Collection of soil and water samples from LA River

June 20

Lab walkthrough
Setting up lab, stock supplies, preparation of LB-amp plates
Test extraction of DNA from BioBrick wells into cloning plates ([http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_K123001 K123001], [http://partsregistry.org/Part:BBa_K123002 K123002], [http://partsregistry.org/Part:BBa_K123003 K123003])
Initial enrichment cultures of collected samples on minimal media with BPA or 17a-ethynylestradiol

June 21

MoBio Powermax Soil kit extraction of DNA from collected samples
Initial enrichment cultures of samples in minimal media with nonylphenol or DDT
Geneious and primer design tutorial

Results

MoBio Extraction

Sample Concentration (ng/µl)
1
4.5
2
4.1
4
0.0
5
0.7
6
13.7
7
5.8
9
58.7
10
11.4

Locations 3 and 8 were omitted from the MoBio Powermax Soil Extraction procedure because they were liquid samples.

June 22

Miniprep and sequencing of selected BioBricks (BBa_K123000, BBa_K123001, BBa_K123002, BBa_K123003)
Preparation of competent cells

Results of OD measurements:

Part Concentration (ng/µl)
K123000
326.6
K123001
293.8
K123002
152.3
K123003
402.6

June 23

Transformation of T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]]), and double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) biobricks for test sequences for BisA and BisB degradation genes ([http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001])
Retested competent cells made June 22nd with [http://partsregistry.org/Part:BBa_K123001 K123001] and pUC
Transfered 0.5 mL aliquots of BPA and 5mL 17a-ethynylestradiol cultures from June 20 to new tubes of minimal media
Designed primers for test sequence of BisdA (pNT001)
Designed primers to continue sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003])

We determined that sequences for [http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001] have correct protein coding but different codons than in the sequences shown online

June 24

Transformation of Tet repressible promoter [http://partsregistry.org/Part:BBa_R0040 R0040]
Preparation of LB, LB (no antibiotic) plates, and SOC media
Transfer of 5 mL DDT and nonylphenol cultures from June 21 to new tubes of minimal media
Design of reverse primer for continued sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003])
Update Wiki with protocols we have been using

We determined that our competent cells are functional have a cfu/ug DNA of 6.6 x 10^6.

June 25

Take out plates from June 24


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