User:University of Washington/22 July 2008

From 2008.igem.org

(Difference between revisions)
(→LuxR from AraC and TetR)
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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>
+
<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>
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*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.

Revision as of 23:29, 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 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.



Team:University_of_Washington/Notebook

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