From 2008.igem.org
| June 18, 2008
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| 1. “Paper Punches”: Transfer DNA/plasmid of chosen gene from iGEM paper into 1.5 ml tube.
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| 2. Assign numbers to genes/plasmids:
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| | *Number* | *Plasmid/gene* | *Resistance type*
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| | 1 | Tet R | Ampicillin Res.
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| | 2 | YFP | Ampicillin Res.
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| | 3 | MCherry | Ampicillin Res.
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| | 4 | P22 MNTR | Kanamycin Res.
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| | 5 | P22 MNTC | Kanamycin Res.
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| | 6 | P22 CIIC | Ampicillin Res.
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| | 7 | LAMBDA CIR | Ampicillin Res.
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| | 8 | EYFP | Ampicillin Res.
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| | 9 | P22 C2R | Ampicillin Res.
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| | 10 | GFP | Ampicillin Res.
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| | 11 | TERMINATOR | Amp. and Kan. Res.
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| | 12 | RBS | Ampicillin Res.
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| | 13 | LAMBDA CIC | Ampicillin Res.
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| | 14 | LAC I | Ampicillin Res.
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| 3. “Transformation”: Transformation means taking the gene/plasmid from the biobrick paper and transforming it to competent cells. Procedure: 2 microliters(uL) of plasmid DNA in TE was added to 50 uL thawed dh5alpha chemocompetent cells in a pre-chilled 1.5 mL tube. Cells were allowed to incubate for approximately 30 minutes on ice; then heat shocked @ 50 Celsius in a H20 bath for 60 seconds with agitation/mixing by flicking tubes @ every 20 second interval. Tubes were placed on ice for 2 minutes, then cells were transferred to 2mililiter glass culture tubes containing 500uL of 2xTy broth pre-warmed @ 37 Celsius. Cells were allowed to recover @ 37 Celsius with shaking @ 225 rpm (rotation-per-minute) for 2 hours. After this, cells were plated on LB plates containing antibiotic (ampicillin or kanamycin; determined by resistance gene in plasmid). Plates were incubated overnight for approximately 14 hours.
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| 4. Steps of making antibiotic LB plate: spray hood area clean w/ 80% ethanol turn on vent in hood so microbes are sucked up and away from plates light a fire to kill airborne bacteria using a pipet, measure ___ of antibiotic into liquid LB, mix, and eye ball pouring use extra safety measures to prevent bacterial contamination since LB is nutrient rich.
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| 5. General lab steps went through for the day: Cut out DNA paper from “Biobricks” section of iGEM notebook (sterilize scalpel by dipping into 10% bleach, water, then ethanol) Place papers in individual 1.5 mL tube Add 5uL of warmed TE for 20 minutes Add 2uL of DNA in TE and 50uL of thawed dh5alpha competent cells Allows DNA to enter competent cells (ice & heat) transfer competent cells w/ DNA to 2xTy broth 500uL 2xTy broth used each placed @ 37C Make agar/LB plate by mixing liquid LB w/ antibiotic (do this so no other cells can grow and contaminate the plate – only competent cells w/ biobrick DNA grows).
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| 6. Biobrick numbers for genes/plasmids:
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| | Gene | Biobrick
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| | GFP | E0040
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| | RFP | J04051
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| | RBS | B0032
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| | Terminator | B1006
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| | Lambda CI | R0051, C0051
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| | P22 CII | R0053, C0053
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| | P22 mnt | R0072, C0072
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| | Tet R | R0040
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| | Lac I | R0010
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|** Biobrick website: parts.mit.edu ***
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