User:University of Washington/25 July 2008

From 2008.igem.org

(Difference between revisions)
(→LuxR from AraC and TetR)
 
(19 intermediate revisions not shown)
Line 1: Line 1:
 +
==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
 +
<table>
 +
<tr>
 +
  <th>reagents</th><th>amount per reaction(ul)</th>
 +
</tr>
 +
<tr>
 +
  <td>sterile dH2O</td><td align=center>34.5</td>
 +
</tr>
 +
<tr>
 +
  <td>buffer for Taq (-MgCl2)</td><td align=center>5</td>
 +
</tr>
 +
<tr>
 +
  <td>10 mM dNTPs</td><td align=center>1</td>
 +
</tr>
 +
<tr>
 +
  <td>MgCl2</td><td align=center>2</td>
 +
</tr>
 +
<tr>
 +
  <td>F primer(#436)</td><td align=center>2.5</td>
 +
</tr>
 +
<tr>
 +
  <td>R primer(#436)</td><td align=center>2.5</td>
 +
</tr>
 +
<tr>
 +
  <td>Taq</td><td align=center>0.5</td>
 +
</tr>
 +
<tr>
 +
  <td>DNA template</td><td align=center>2</td>
 +
</tr>
 +
</table>
 +
*Thermocycling
 +
<table>
 +
<tr>
 +
  <th>Segment</th><th>Cycles</th><th>Temperature</th><th>Time</th>
 +
</tr>
 +
<tr align=center>
 +
  <td>1</td><td>1</td><td>95°C</td><td>1 minute</td>
 +
</tr>
 +
<tr align=center>
 +
  <td rowspan=3 valign=top>2</td><td rowspan=3 valign=top>30</td><td>95°C</td><td>30 seconds</td>
 +
</tr>
 +
<tr align=center>
 +
  <td>50°C</td><td>1 min</td>
 +
</tr>
 +
<tr align=center>
 +
  <td>72°C</td><td>1 min</td>
 +
</tr>
 +
<tr align=center>
 +
  <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>
 +
</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
reagentsamount per reaction(ul)
sterile dH2O34.5
buffer for Taq (-MgCl2)5
10 mM dNTPs1
MgCl22
F primer(#436)2.5
R primer(#436)2.5
Taq0.5
DNA template2
  • Thermocycling
SegmentCyclesTemperatureTime
1195°C1 minute
23095°C30 seconds
50°C1 min
72°C1 min
3172°C7 minutes
4-4°Cinfinite

- 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

Retrieved from "http://2008.igem.org/User:University_of_Washington/25_July_2008"