Team:Caltech/Week 8

From 2011.igem.org

(Difference between revisions)
Line 38: Line 38:
Gibson assemble pNT002 using the gel extracted insert and linearized pSB3K3<br/>
Gibson assemble pNT002 using the gel extracted insert and linearized pSB3K3<br/>
Gibson assemble pNT003 using the gel extracted insert and linearized pSB4A5<br/>
Gibson assemble pNT003 using the gel extracted insert and linearized pSB4A5<br/>
 +
Transform Gibson reactions into our XL-10 cells and Emzo's top ten cells<br/>
Replating of more pUC19-transformed competent cells and non-transformed competent cells<br/>
Replating of more pUC19-transformed competent cells and non-transformed competent cells<br/>
Standard Assembly Digest- K12300, B1004, R0010, B0034, along with the destination plasmids.<br/>  
Standard Assembly Digest- K12300, B1004, R0010, B0034, along with the destination plasmids.<br/>  
Line 47: Line 48:
File:8-2pcrgel2.jpg|lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5-7 pNT003 inset with PIPE primers, 8-9 pNT003 insert, 10-11 pNT003 inset with PIPE primers, 12-15 pETDEST53
File:8-2pcrgel2.jpg|lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5-7 pNT003 inset with PIPE primers, 8-9 pNT003 insert, 10-11 pNT003 inset with PIPE primers, 12-15 pETDEST53
</gallery>
</gallery>
-
pNT002 ligation showed band on gel
+
<p>pNT002 ligation showed band on gel</p>
 +
pSB3K3 concentration: 90.9 ng/ul
 +
==August 3==
 +
Colony PCR of Gibson transforms<br/>
 +
===Results===
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>number of colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pNT002 + top ten</td>
 +
<td>55</td>
 +
</tr>
 +
<tr>
 +
<td>pNT002 - top ten</td>
 +
<td>43</td>
 +
<tr>
 +
<td>pNT002 +XL10 gold</td>
 +
<td>180</td>
 +
</tr>
 +
<tr>
 +
<td>pNT002 - XL10 gold</td>
 +
<td>400</td>
 +
</tr>
 +
<td>pNT003 + top ten</td>
 +
<td>14</td>
 +
</tr>
 +
<tr>
 +
<td>pNT003 - top ten</td>
 +
<td>1</td>
 +
<tr>
 +
<td>pNT003 +XL10 gold</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT003 - XL10 gold</td>
 +
<td>7</td>
 +
</tr>
 +
</table>
}}
}}

Revision as of 18:34, 3 August 2011


Caltech iGEM 2011



Home

Project

Data

Parts

Team

Notebook

Biosafety

Human Impact

References

Support

July 31

Started a LB+amp overnight culture of the B0034 glycerol stock from the 2010 Caltech iGEM Team

Results

Mass spectrometry did not run correctly; samples did not ionize; must rerun

August 1

PCR pSB3K3 and pNT003 insert with the new primers.
Gel extract the pNT002 insert from the July 29 PCR
Minimal media transfers
Transformation and plating of pUC19 into competent cells for cell dry weight experiment
Overnight culture of B0034 glycerol stock
Overnight ligation of pNT002 insert and PSB3K3 backbone
Restriction digest of parts for pNT002 standard assembly and overnight ligation

Results

No colonies visible on the pUC19-transformed cells

The enrichment culture plates show no visible growth yet.

Gel Extractions of July 29 PCR of pNT002 and pNT003 inserts

Part Concentration (ng/ul)
pNT002 insert 65.9
pNt003 insert 12.5

August 2

Gibson assemble pNT002 using the gel extracted insert and linearized pSB3K3
Gibson assemble pNT003 using the gel extracted insert and linearized pSB4A5
Transform Gibson reactions into our XL-10 cells and Emzo's top ten cells
Replating of more pUC19-transformed competent cells and non-transformed competent cells
Standard Assembly Digest- K12300, B1004, R0010, B0034, along with the destination plasmids.
Run gel of overnight pNT002 ligations
Transformed pNT002 insert+ pSB3K3 ligation

Results

lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5-7 pNT003 insert with PIPE primers, 8-13 pSB3K3, 14-15 pETDEST53

lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5-7 pNT003 inset with PIPE primers, 8-9 pNT003 insert, 10-11 pNT003 inset with PIPE primers, 12-15 pETDEST53

pNT002 ligation showed band on gel

pSB3K3 concentration: 90.9 ng/ul

August 3

Colony PCR of Gibson transforms

Results

</tr>

Plate number of colonies
pNT002 + top ten 55
pNT002 - top ten 43
pNT002 +XL10 gold 180
pNT002 - XL10 gold 400
pNT003 + top ten 14
pNT003 - top ten 1
pNT003 +XL10 gold 0
pNT003 - XL10 gold 7


Retrieved from "http://2011.igem.org/Team:Caltech/Week_8"