User:University of Washington/22 July 2008

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(→RP4 Conjugation)
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[[UW_Glycerol_Stocks| Glycerol Stock]] DH5alpha RP4
[[UW_Glycerol_Stocks| Glycerol Stock]] DH5alpha RP4
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==LuxR from AraC and TetR==
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- QuikChange Mutagenesis
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<table>
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<tr>
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<th>Materials</th><th>Reaction#1</th><th>Reaction#2</th><th>negative</th><th>positive</th>
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</tr>
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<tr>
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<td>AraC plasmid</td><td>39.4 ul</td><td>39.5 ul</td><td>39.4 ul</td><td>5 ul(only had that much left)</td>
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</tr>
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<tr>
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<td>10X pfuTurbo reaction buffer</td><td>5 ul</td><td>5 ul</td><td>5 ul</td><td>5 ul</td>
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</tr>
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<tr>
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<td>dNTP mix</td><td>1 ul</td><td>1 ul</td><td>1 ul</td><td>1 ul</td>
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</tr>
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<tr>
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<td>primer AraC-F</td><td>0.806 ul AraC1F</td><td>0.728 ul AraC2F</td><td>-</td><td>0.806 ul AraC1F</td>
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</tr>
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<tr>
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<td>primer AraC-R</td><td>0.806 ul AraC1R</td><td>0.728 ul AraC2R</td><td>-</td><td>0.806 ul AraC1R</td>
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</tr>
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<tr>
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<td>DMSO</td><td>3 ul</td><td>3 ul</td><td>3 ul</td><td>3 ul</td>
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</tr>
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<tr>
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<td>ddH2O</td><td>-</td><td>-</td><td>1.6 ul</td><td>34.4 ul</td>
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</tr>
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</table>
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*add 1 ul pfuTurbo in both
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*Temperature Cycle(There are small changes from our [[Team:University_of_Washington/Protocols|protocol page]].
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<table>
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<tr>
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  <th>Segment</th><th>Cycles</th><th>Temperature</th><th>Time</th>
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</tr>
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<tr>
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  <td>1</td><td>1</td><td>95°C</td><td><font color="red">2 minute</font></td>
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</tr>
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<tr>
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  <td rowspan=3>2</td><td rowspan=3>18</td><td>95°C</td><td>50 seconds</td>
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</tr>
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<tr>
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  <td>60°C</td><td>50 seconds</td>
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</tr>
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<tr>
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  <td>68°C</td><td><font color="red">3 mins</font></td>
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</tr>
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<tr>
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  <td>3</td><td>1</td><td>68°C</td><td>7 minutes</td>
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</tr>
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<tr>
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  <td rowspan="3">let it sit in 4°C</td>
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</tr>
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</table>
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Revision as of 23:27, 22 July 2008

RP4 Conjugation

Test conjugation protocol #2
·DH5alpha RP4 + pCS26-pac
·DH5alpha + ""

Glycerol Stock DH5alpha RP4

LuxR from AraC and TetR

- QuikChange Mutagenesis

MaterialsReaction#1Reaction#2negativepositive
AraC plasmid39.4 ul39.5 ul39.4 ul5 ul(only had that much left)
10X pfuTurbo reaction buffer5 ul5 ul5 ul5 ul
dNTP mix1 ul1 ul1 ul1 ul
primer AraC-F0.806 ul AraC1F0.728 ul AraC2F-0.806 ul AraC1F
primer AraC-R0.806 ul AraC1R0.728 ul AraC2R-0.806 ul AraC1R
DMSO3 ul3 ul3 ul3 ul
ddH2O--1.6 ul34.4 ul
  • add 1 ul pfuTurbo in both
  • Temperature Cycle(There are small changes from our protocol page.
SegmentCyclesTemperatureTime
1195°C2 minute
21895°C50 seconds
60°C50 seconds
68°C3 mins
3168°C7 minutes
let it sit in 4°C



Team:University_of_Washington/Notebook

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