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User:University of Washington/10 July 2008
From 2008.igem.org
Contents |
RP4 Conjugation
Conjugation protocol #1
·DH5α
·DH5α + S17-1 w/ RP4
·DH5α + naked RP4 DNA
·DH5α + amp resistant RFP plasmid
Lambda Red
·Synthesis tetR insert for KorA and trbA replacement
non-RP4 Conjugation
Mini Prep & Glycerol Stock
·CV13(Yep13)
·pDPT51
LuxR from AraC and TetR
- GFP E0240 grew, got some single colonies, stored in a fridge.
- Finished up QuikChange Mutagenesis: Dpn1 digestion.
- Did PCR purification with 30 ul EB Buffer.
- Transformed mutated AraC plasmids into XL1 Blue cells.
//Note: Electroporator 200 Ohms, 1.5 kV, with Tsy Media
//Note: Failed to put cells on ice while bringing them over to do the electroporation. Hopefully, they will still grow.
- Tube 1: 45 ul XL1 Blue + 15 ul DNA#1
- Tube 2: 45 ul XL1 Blue + 2 ul DNA#1
- Tube 3: 45 ul XL1 Blue + 15 ul DNA#2
- Tube 4: 45 ul XL1 Blue + 2 ul DNA#2
- Tube 5: 45 ul XL1 Blue
- Plated 200 ul each on LB agar + Amp
BioBrick Promoter Measurements
- Fluorescence and OD600 data was retrieved for the promoter-construct transformants and control groups. The data was manipulated and interpreted in MATLAB. A slope comparison method and a normalized-fluorescence method for singular log-phase data points were used to determine promoter strengths. The slope and data points associated with the empty TOP10 cells were accounted for. The promoter strengths gleaned from the slope-comparison method, the normalized-fluorescence method, and the values listed in the Measurement Kit are provided, respectively, below:
-I20260 (promoter J23101): 1, 1, 1
-I20268 (promoter J23102): 1.09, 0.97, 0.96
-I20269 (promoter J23150): 0.19, 0.24, 0.26
-I20270 (promoter J23151): 0.44, 0.57, 0.55
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