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User:University of Washington/10 July 2008
From 2008.igem.org
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·Synthesis tetR insert for KorA and trbA replacement | ·Synthesis tetR insert for KorA and trbA replacement | ||
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| + | == Lamdba Red Recombineering of RP4 (Bryan) == | ||
| + | |||
| + | Attemped to PCR amplify recombinant fragments containing selectable cassette. Probable source of error: wrong PCR reaction buffer or primer dilution. | ||
| + | |||
| + | Chemical transformation of C0161 LuxI gene into Top10 failed. | ||
== non-RP4 Conjugation == | == non-RP4 Conjugation == | ||
Revision as of 00:39, 12 July 2008
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
Lamdba Red Recombineering of RP4 (Bryan)
Attemped to PCR amplify recombinant fragments containing selectable cassette. Probable source of error: wrong PCR reaction buffer or primer dilution.
Chemical transformation of C0161 LuxI gene into Top10 failed.
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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