User:University of Washington/22 July 2008

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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