User:University of Washington/22 July 2008

From 2008.igem.org

(Difference between revisions)
(LuxR from AraC and TetR)
(LuxR from AraC and TetR)
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- added Dpn1 in each tube EXCEPT positive control, let the reaction sit in 37 degree Celsius.
- 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.

Revision as of 01:37, 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.



Team:University_of_Washington/Notebook