Team:UC Davis/Protocols

From 2013.igem.org

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      <h1 class="title">Protocols</h1>
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      <h2 class="title">LB Media</h2>
 +
<li>950 mL  H<sub>2</sub>0</li>
 +
<li>10 g      Tryptone</li>
 +
<li>10 g      NaCl</li>
 +
<ins><li>5 g        Yeast Extract</li></ins>
 +
<li>1 L  Total</li>
 +
<li>Add 15 g Agar, if being poured into plates.</li>
 +
<li>Autoclave, when cool add antibiotics if desired.</li>
 +
      <h2 class="title">Antibiotic Stock Solutions</h2>
 +
Chloramphenicol
 +
<li>Working Concentration 12.5 μg/ml</li>
 +
<li>Stock solutions can be made at 35 mg/ml in ethanol and kept at -20º C</li>
 +
<br><br />
 +
Kanamycin
 +
<li>Filter sterilize for kanamycin</li>
 +
Working Concentration:
 +
<li>25 μg/mL for low-copy plasmids</li>
 +
<li>35 µg/mL for high-copy plasmids</li>
 +
<li>Stock solution is 35 mg/ml in water and kept at -20º C</li>
 +
<br><br />
 +
Spectinomycin
 +
<li>Filter sterilize</li>
 +
<li>Working Concentration 50 μg/mL</li>
 +
<li>Stock solution is 100 mg/mL in water and kept at -20º C</li>
 +
      <h2 class="title">Heat Shock Transformation</h2>
 +
<ol>
 +
<li>Preheat water bath to 42º C.</li>
 +
<li>Thaw competent cells on ice for 10 minutes.</li>
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<li>Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes. </li>
 +
<li>Heat shock in 42º C water bath for 45 seconds.</li>
 +
<li>Cool cells in ice bath for a few minutes.</li>
 +
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li>
 +
<li>Transfer 200 µL of culture per plate (containing the appropriate antibiotic).</li>
 +
<li>Spread using glass beads.</li>
 +
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.< /li>
 +
</ol>
 +
      <h2 class="title">Making Chemically Competent Cells</h2>
 +
      <h2 class="title">Double Restriction (Fast) Digest </h2>
 +
      <h2 class="title">Ligations (Standard Assembly)</h2>
 +
      <h2 class="title">Gel Electrophoresis</h2>
 +
      <h2 class="title">Gel Extraction and Purification</h2>
 +
      <h2 class="title">PCR Amplification for Golden Gate Assembly</h2>
 +
      <h2 class="title">Golden Gate Assembly</h2>
 +
      <h2 class="title">DNA Extraction (Minipreps)</h2>
 +
      <h2 class="title">Making Glycerol Stocks</h2>
 +
      <h2 class="title">Sequencing Prep</h2>
 +
      <h2 class="title">Tecan Testing Protocol</h2>
 +
      <h2 class="title">Site-Directed Mutagenesis PCR</h2>
 +
      <h2 class="title">Electroporation Transformation</h2>

Revision as of 18:32, 15 August 2013

Protocols

LB Media

  • 950 mL H20
  • 10 g Tryptone
  • 10 g NaCl
  • 5 g Yeast Extract
    •
  • 1 L Total
  • Add 15 g Agar, if being poured into plates.
  • Autoclave, when cool add antibiotics if desired.

    • Antibiotic Stock Solutions

    Chloramphenicol
  • Working Concentration 12.5 μg/ml
  • Stock solutions can be made at 35 mg/ml in ethanol and kept at -20º C


    • Kanamycin
  • Filter sterilize for kanamycin
    • Working Concentration:
  • 25 μg/mL for low-copy plasmids
  • 35 µg/mL for high-copy plasmids
  • Stock solution is 35 mg/ml in water and kept at -20º C


    • Spectinomycin
  • Filter sterilize
  • Working Concentration 50 μg/mL
  • Stock solution is 100 mg/mL in water and kept at -20º C

    • Heat Shock Transformation

    1. Preheat water bath to 42º C.
    2. Thaw competent cells on ice for 10 minutes.
    3. Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes.
    4. Heat shock in 42º C water bath for 45 seconds.
    5. Cool cells in ice bath for a few minutes.
    6. Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes.
    7. Transfer 200 µL of culture per plate (containing the appropriate antibiotic).
    8. Spread using glass beads.
    9. Invert plates and incubate overnight (12-16 hrs) at 37º C.< /li>

    Making Chemically Competent Cells

    Double Restriction (Fast) Digest

    Ligations (Standard Assembly)

    Gel Electrophoresis

    Gel Extraction and Purification

    PCR Amplification for Golden Gate Assembly

    Golden Gate Assembly

    DNA Extraction (Minipreps)

    Making Glycerol Stocks

    Sequencing Prep

    Tecan Testing Protocol

    Site-Directed Mutagenesis PCR

    Electroporation Transformation

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