Team:Caltech/Week 3

From 2011.igem.org

(Difference between revisions)
Line 50: Line 50:
==June 28==
==June 28==
-
<p>Send off continued forward sequencing for HER <br>
+
<p>Send off continued forward sequencing for HER<br>
-
Prepare gel of control fosmid DNA to practice gel extraction technique<br/>
+
PCR for Gibson assembly of PNT001 and PNT002<br/>
-
PCR for Gibson<br/>
+
Gel and PCR purification of PCR products <br/>
-
Gibson assembly of pNT001 and pNT002</p>
+
Analysis of sequencing results from yesterday<br/>
 +
Transfer DDT and nonylphenol cultures to new media <br/>
 +
Transform 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])for creation of glycerol stocks
 +
 
===Results===
===Results===
 +
Sequencing: All biobricks showed correct sequence except T7 Polymerase
[[File:628pcrgel.png|thumb|PCR of parts for pNT001 and pNT002: From left to right: 1 ladder; 2 blank; 3 [http://partsregistry.org/Part:BBa_R0010 R0010]; 4 [http://partsregistry.org/Part:BBa_K123000 K123000]; 5 [http://partsregistry.org/Part:BBa_B0014 B0014]; 6 [http://partsregistry.org/Part:BBa_R0040 R0040]; 7 [http://partsregistry.org/Part:BBa_K123001 K123001]; 8 [http://partsregistry.org/Part:BBa_B0015 B0015] ]]
[[File:628pcrgel.png|thumb|PCR of parts for pNT001 and pNT002: From left to right: 1 ladder; 2 blank; 3 [http://partsregistry.org/Part:BBa_R0010 R0010]; 4 [http://partsregistry.org/Part:BBa_K123000 K123000]; 5 [http://partsregistry.org/Part:BBa_B0014 B0014]; 6 [http://partsregistry.org/Part:BBa_R0040 R0040]; 7 [http://partsregistry.org/Part:BBa_K123001 K123001]; 8 [http://partsregistry.org/Part:BBa_B0015 B0015] ]]
Ran a gel of the PCR products. Strangely, lanes 3 and 4 show no DNA. We will repeat the PCR of lanes 3-5 and Gibson assemble pNT001 ([http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_B0014 B0014]) later in the week.<br/><br/>
Ran a gel of the PCR products. Strangely, lanes 3 and 4 show no DNA. We will repeat the PCR of lanes 3-5 and Gibson assemble pNT001 ([http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_B0014 B0014]) later in the week.<br/><br/>
Line 95: Line 99:
<td>74.0</td>
<td>74.0</td>
</tr>
</tr>
-
</table>
+
</table> </p>
 +
==June 29==
 +
<p>
 +
Redo PCR of [http://partsregistry.org/Part:BBa_R0010 R0010] and [http://partsregistry.org/Part:BBa_K123000 K123000]<br/>
 +
Gibson assemble pNT002, pNT001 if redo of PCR works<br/>
 +
Order primers and vectors for 16s sequencing<br/>
 +
Analyze sequence of ER, design test plasmids if sequence of ER is correct, if not, plan repair <br/>
 +
Plan experiments using pNT001 and pNT002</p>
 +
 
== This Week ==
== This Week ==
-
<p>PCR of parts for pNT001 (Test plasmid for [http://partsregistry.org/Part:BBa_K123000 K123000])<br/>
+
<p>
-
Purification/Gibson assembly for pNT001<br/>
+
Email other teams <br/>
Email other teams <br/>
-
PCR of parts for pNT002 and assembly<br/>
 
Use pNT001 and pNT002 to test degradation parts</p>
Use pNT001 and pNT002 to test degradation parts</p>
}}
}}

Revision as of 19:09, 29 June 2011


Caltech iGEM 2011



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Data

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Team

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Human Impact

References

Support

June 26

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])

June 27

Miniprep 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]) and submit for sequencing
Transfer 0.5 mL aliquots of BPA and 5 mL aliquots 17a-ethynylestradiol cultures from June 23 to fresh minimal media
Checked DDT and nonylphenol cultures, will wait until tomorrow to transfer
Ethanol precipitation of DNA extractions from LA river samples following protocol
Streak out cells from fosmid kit
Prepare antibiotic stocks (Ampicillin, Chloramphenicol)

Results

Miniprep

Part Concentration(ng/ul)
B0014 230.5
B0015 254.1
J06702 301.3
K145001 205.6
R0010 117.3
R0040 156.8

We found growth in one of our BPA enrichment cultures, and slight growth in our ethinyl estradiol cultures. We transferred these vials today.

June 28

Send off continued forward sequencing for HER
PCR for Gibson assembly of PNT001 and PNT002
Gel and PCR purification of PCR products
Analysis of sequencing results from yesterday
Transfer DDT and nonylphenol cultures to new media
Transform 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])for creation of glycerol stocks

Results

Sequencing: All biobricks showed correct sequence except T7 Polymerase

PCR of parts for pNT001 and pNT002: From left to right: 1 ladder; 2 blank; 3 [http://partsregistry.org/Part:BBa_R0010 R0010]; 4 [http://partsregistry.org/Part:BBa_K123000 K123000]; 5 [http://partsregistry.org/Part:BBa_B0014 B0014]; 6 [http://partsregistry.org/Part:BBa_R0040 R0040]; 7 [http://partsregistry.org/Part:BBa_K123001 K123001]; 8 [http://partsregistry.org/Part:BBa_B0015 B0015]

Ran a gel of the PCR products. Strangely, lanes 3 and 4 show no DNA. We will repeat the PCR of lanes 3-5 and Gibson assemble pNT001 ([http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_B0014 B0014]) later in the week.

EtOH precipitation of Soil Extractions from June 21

Tube/Location Number Concentration(ng/ul)
1 13.6
2 15.2
4 8.3
5 11.4
6 25.5
7 23.5
9 261.3
10 74.0

June 29

Redo PCR of [http://partsregistry.org/Part:BBa_R0010 R0010] and [http://partsregistry.org/Part:BBa_K123000 K123000]
Gibson assemble pNT002, pNT001 if redo of PCR works
Order primers and vectors for 16s sequencing
Analyze sequence of ER, design test plasmids if sequence of ER is correct, if not, plan repair
Plan experiments using pNT001 and pNT002

This Week

Email other teams
Use pNT001 and pNT002 to test degradation parts


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