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User:University of Washington/14 August 2008
From 2008.igem.org
LuxR from AraC and TetR(Faifan)
QuikChange Approach
- miniprepped QuikChange overnight cultures (rxn1 x 4, rxn2) and submit sequencing:Forward
Elowitz
-dilute 1:10 of digested BBa_Elowitz
-3 way ligation(20 ul rxn)
- mix
| dH20(ul) | Buffer(ul) | P1010-Kan(ul) | BBa_Elowitz(ul) | GFP(ul) | LuxR(ul) | T4 ligase(ul) | |
|---|---|---|---|---|---|---|---|
| GFP | 14.6 | 2 | 0.40 | 1 | 1 | - | 1>/td> |
| LuxR | 14.6 | 2 | 0.40 | 1 | - | 1 | 1>/td> |
- 30 mins incubate, room temp
- 10 mins denature, 65 degree Celsius
- 20 mins filtration
-transformed into XL1-Blue and plated on Kan
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