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User:University of Washington/13 August 2008
From 2008.igem.org
Contents |
Conjugation (Scott)
experiment from Aug. 7 results
| experiment # | Bacteria | Yeast | Yeast count | transconjugation efficiency |
|---|---|---|---|---|
| 1 | 250uL | 250uL | 66 | 2.64E-5 |
| 2 | 200uL | 300uL | 64 | 2.13E-5 |
| 3 | 100uL | 400uL | 32 | 8.00e-6 |
| 4 | 50uL | 450uL | 18 | 4.00e-6 |
LuxR from AraC and TetR(Faifan)
QuikChange Approach
- Overnight QuikChange#4 culture from Amp plate (reaction1 x 4, reaction2 x 2) in Tsy+Amp
- Sequence of DNA from QuikChange#3 came back ==> No mutation.
Elowitz
-Made Glycerol Stock of BBa_Elowitz x 2
-Restriction Digest of BBa_Elowitz, BBa_E0240(GFP), BBa_I0462(LuxR), BBa_P1010(on pSB3K3)
- mix
| dH2O(ul) | Buffer(ul) | BSA(ul) | DNA(ul) | E(ul) | X(ul) | S(ul) | P(ul) | |
|---|---|---|---|---|---|---|---|---|
| BBa_Elowitz | 32.5 | EcoRI, 5 | 0.5 | 10 | 1 | - | 1 | - |
| LuxR | 32.5 | NEB3, 5 | 0.5 | 10 | - | 1 | - | 1 |
| GFP | 32.5 | NEB3, 5 | 0.5 | 10 | - | 1 | - | 1 |
| P1010 | 32.5 | EcoRI, 5 | 0.5 | 10 | 1 | - | - | 1 |
- incubate 37 degree Celsius, 1.5 hours
- 15 mins water bath 80 degree Celsius
- nanodrop at 230 nm
| ng/ul | 260/280 | 260/230 | |
|---|---|---|---|
| BBa_Elowitz | 76.2 | 1.12 | 0.34 |
| LuxR | 43.5 | 0.98 | 0.52 |
| GFP | 40.7 | 1.00 | 0.46 |
| P1010 | 50.8 | 0.78 | 0.17 |
- store in 4 degree Celsius
Lambda Red Recombineering of RP4 (Bryan)
Attempted the following transformations RP1 + pKD78, RP4 + pKD78, and pKD78 alone. Selected with cam/tet, cam/tet, and cam, resepectively. None of the O.N. cultures grew. Possible problems are imprecision in micropipetting due to extremely small volumes <0.5 ul of vector DNA or not enough cells (1 mL of culture per electroporation).
AHL Expression in Yeast (Bryan)
Transformed C0161/YEp426 ligation produce from yeseterday into XL-1 Blue. O.N. culture grew in amp, indicating successful transformation. Miniprepped and made glycerol stock.
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