Minnesota/18 June 2008

From 2008.igem.org

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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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|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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|2. Assign numbers to genes/plasmids:
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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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|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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|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 @ 37C  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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|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 @ 37C  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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|6. Biobrick numbers for genes/plasmids:  
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Revision as of 15:01, 27 June 2008

June 18, 2008
1. “Paper Punches”: Transfer DNA/plasmid of chosen gene from iGEM paper into 1.5 ml tube.
2. Assign numbers to genes/plasmids:
Number Plasmid/gene Resistance type
1 Tet R Ampicillin Res.
2 YFP Ampicillin Res.
3 MCherry Ampicillin Res.
4 P22 MNTR Kanamycin Res.
5 P22 MNTC Kanamycin Res.
6 P22 CIIC Ampicillin Res.
7 LAMBDA CIR Ampicillin Res.
8 EYFP Ampicillin Res.
9 P22 C2R Ampicillin Res.
10 GFP Ampicillin Res.
11 TERMINATOR Amp. and Kan. Res.
12 RBS Ampicillin Res.
13 LAMBDA CIC Ampicillin Res.
14 LAC I Ampicillin Res.
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.
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.
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 @ 37C  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).
6. Biobrick numbers for genes/plasmids:
Gene Biobrick
GFP E0040
RFP J04051
RBS B0032
Terminator B1006
Lambda CI R0051, C0051
P22 CII R0053, C0053
P22 mnt R0072, C0072
Tet R R0040
Lac I R0010
    • Biobrick website: parts.mit.edu ***
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