User:University of Washington/22 July 2008

From 2008.igem.org

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==LuxR from AraC and TetR==
==LuxR from AraC and TetR==
 +
 +
- Made 6 overnight cultures for AraC plasmid (R0080).
- QuikChange Mutagenesis
- QuikChange Mutagenesis
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</tr>
</tr>
<tr>
<tr>
-
<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>
+
<td>primer AraC-F</td><td>0.806 ul 1F</td><td>0.728 ul 2F</td><td>-</td><td>0.806 ul 1F</td>
</tr>
</tr>
<tr>
<tr>
-
<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>
+
<td>primer AraC-R</td><td>0.806 ul 1R</td><td>0.728 ul 2R</td><td>-</td><td>0.806 ul 1R</td>
</tr>
</tr>
<tr>
<tr>
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</tr>
</tr>
</table>
</table>
-
*add 1 ul pfuTurbo in both
+
*add 1 ul pfuTurbo in each tubes
-
*Temperature Cycle(There are small changes from our [[Team:University_of_Washington/Protocols|protocol page]].
+
*Temperature Cycle(There are small changes from our [[Team:University_of_Washington/Protocols|protocol page]]).
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 +
- added Dpn1 in each tube EXCEPT positive control, let the reaction sit in 37 degree Celsius.
 +
 +
- After 2.5 hours, the DNA product was taken out to do PRC purification. (30 EB Buffer at the end)
 +
 +
- Electroporated the purified DNA into XL1-Blue. 45 ul cells + 2 ul DNA (4 tubes)
 +
 +
- Let it sit 30 mins and then inoculated onto LB Agar + Amp plate. Stored in 37 degree Celsius incubator.
 +
==Lambda Red Recombineering of RP4 ==
 +
Troubleshooting of PCR continued.  Performed 2 sets of reactions in parallel -- one using the iGEM lab's reagents and the other using grad advisor's reagents.  My colony PCR failed, while Ingrid's was successful.  Both of our control reactions on confirmed part J04430 were successful, though Ingrid got better yield.  Also got amplification of C0161.  Process of elimination leads me to believe that magnesium chloride concentration is not optimized.
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[[Team:University_of_Washington/Notebook]]
[[Team:University_of_Washington/Notebook]]

Latest revision as of 22:59, 23 July 2008

RP4 Conjugation

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

Glycerol Stock DH5alpha RP4

LuxR from AraC and TetR

- Made 6 overnight cultures for AraC plasmid (R0080).

- 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 1F0.728 ul 2F-0.806 ul 1F
primer AraC-R0.806 ul 1R0.728 ul 2R-0.806 ul 1R
DMSO3 ul3 ul3 ul3 ul
ddH2O--1.6 ul34.4 ul
  • add 1 ul pfuTurbo in each tubes
  • 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

- added Dpn1 in each tube EXCEPT positive control, let the reaction sit in 37 degree Celsius.

- After 2.5 hours, the DNA product was taken out to do PRC purification. (30 EB Buffer at the end)

- Electroporated the purified DNA into XL1-Blue. 45 ul cells + 2 ul DNA (4 tubes)

- Let it sit 30 mins and then inoculated onto LB Agar + Amp plate. Stored in 37 degree Celsius incubator.

Lambda Red Recombineering of RP4

Troubleshooting of PCR continued. Performed 2 sets of reactions in parallel -- one using the iGEM lab's reagents and the other using grad advisor's reagents. My colony PCR failed, while Ingrid's was successful. Both of our control reactions on confirmed part J04430 were successful, though Ingrid got better yield. Also got amplification of C0161. Process of elimination leads me to believe that magnesium chloride concentration is not optimized.


Team:University_of_Washington/Notebook