User:University of Washington/25 July 2008
From 2008.igem.org
(Difference between revisions)
(→LuxR from AraC and TetR) |
(→LuxR from AraC and TetR) |
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-Received primers to biobrick Elowitz's plasmid. | -Received primers to biobrick Elowitz's plasmid. | ||
- | -Did PCR amplification on Elowitz's plasmid. | + | -Did PCR amplification on Elowitz's plasmid (from 4 minipreps). |
*Reaction Set-Up | *Reaction Set-Up | ||
Line 36: | Line 36: | ||
</table> | </table> | ||
*Thermocycling | *Thermocycling | ||
- | <table | + | <table> |
<tr> | <tr> | ||
<th>Segment</th><th>Cycles</th><th>Temperature</th><th>Time</th> | <th>Segment</th><th>Cycles</th><th>Temperature</th><th>Time</th> | ||
</tr> | </tr> | ||
- | <tr> | + | <tr align=center> |
<td>1</td><td>1</td><td>95°C</td><td>1 minute</td> | <td>1</td><td>1</td><td>95°C</td><td>1 minute</td> | ||
</tr> | </tr> | ||
- | <tr> | + | <tr align=center> |
- | <td rowspan=3>2</td><td rowspan=3> | + | <td rowspan=3 valign=top>2</td><td rowspan=3 valign=top>30</td><td>95°C</td><td>30 seconds</td> |
</tr> | </tr> | ||
- | <tr> | + | <tr align=center> |
- | <td> | + | <td>50°C</td><td>1 min</td> |
</tr> | </tr> | ||
- | <tr> | + | <tr align=center> |
- | <td> | + | <td>72°C</td><td>1 min</td> |
</tr> | </tr> | ||
- | <tr> | + | <tr align=center> |
- | <td>3</td><td>1</td><td> | + | <td>3</td><td>1</td><td>72°C</td><td>7 minutes</td> |
+ | </tr> | ||
+ | <tr align=center> | ||
+ | <td>4</td><td>-</td><td>4°C</td><td>infinite</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | - Good news. Ran gel on PCR product. Expected 172 bp. Got fragments shorter than 500 kb. =] | ||
+ | |||
+ | - Bad news. Sequencing result for second trial Quikchange came. The sequences did not show any mutation. =[ | ||
+ | |||
+ | ==LuxR from pLac== | ||
+ | |||
+ | -R0010+E0420 transformed cells' DNA was sent in for sequencing. | ||
+ | |||
+ | -Glycerol stock of DH5a+lacq strain made. | ||
+ | |||
+ | -One aliquot of electrocompetent DH5a+lacq cells were made. | ||
+ | |||
+ | -Sequence of part I0462 from the 2007 plates was received back and found to be the incorrect sequence. Sent an email to igem hq requesting bacterial stab of part I0462. | ||
+ | |||
---- | ---- | ||
Back to [[Team:University_of_Washington/Notebook#Notebook]] | Back to [[Team:University_of_Washington/Notebook#Notebook]] |
Latest revision as of 00:06, 26 July 2008
LuxR from AraC and TetR
-Received primers to biobrick Elowitz's plasmid.
-Did PCR amplification on Elowitz's plasmid (from 4 minipreps).
- Reaction Set-Up
reagents | amount per reaction(ul) |
---|---|
sterile dH2O | 34.5 |
buffer for Taq (-MgCl2) | 5 |
10 mM dNTPs | 1 |
MgCl2 | 2 |
F primer(#436) | 2.5 |
R primer(#436) | 2.5 |
Taq | 0.5 |
DNA template | 2 |
- Thermocycling
Segment | Cycles | Temperature | Time |
---|---|---|---|
1 | 1 | 95°C | 1 minute |
2 | 30 | 95°C | 30 seconds |
50°C | 1 min | ||
72°C | 1 min | ||
3 | 1 | 72°C | 7 minutes |
4 | - | 4°C | infinite |
- Good news. Ran gel on PCR product. Expected 172 bp. Got fragments shorter than 500 kb. =]
- Bad news. Sequencing result for second trial Quikchange came. The sequences did not show any mutation. =[
LuxR from pLac
-R0010+E0420 transformed cells' DNA was sent in for sequencing.
-Glycerol stock of DH5a+lacq strain made.
-One aliquot of electrocompetent DH5a+lacq cells were made.
-Sequence of part I0462 from the 2007 plates was received back and found to be the incorrect sequence. Sent an email to igem hq requesting bacterial stab of part I0462.
Back to Team:University_of_Washington/Notebook#Notebook