Team:Calgary/Notebook/Protocols/Process5

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This protocol is used to prepare f2media for algae growth</p>
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This protocol is used to prepare f2 media for algal growth.</p>
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<h4> Stock solutions </h4>
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<li>NaNO<sub>3</sub> (150.0 g/L)</li>
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<li>Trace metals (mixed to 750 mL to dissolve, and then brought up to 1 L):  CuSO<sub>4</sub>.5H<sub>2</sub>O (19.6 mg/L), ZnSO<sub>4</sub>.7H<sub>2</sub>O (44.0 mg/L), CoCl<sub>2</sub>.6H<sub>2</sub>O (22.0 mg/L), MnCl<sub>2</sub>.4H<sub>2</sub>O (360.0 mg/L), Na<sub>2</sub>MoO<sub>4</sub>.2H<sub>2</sub>O (12.6 mg/L) </li>
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<li> Na<sub>2</sub>SiO<sub>3</sub>.5H<sub>2</sub>O (22.7 g/L) </li>
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<li>Fe citrate (both constituents added to the same 1 L: Ferric citrate (9.0g/L), Citric acid (9.0g/L)</li>
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<li>Vitamin primary stocks: of Biotin (10.0 mg/100 mL) and vitamin B12 (10.0 mg/100 mL).</li>
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<li>Vitamin working solutions (made fresh every three months) containing: 1.0mL primary stock Biotin, 1.0 mL Vitamin B12, 20.0 mg Thiamine HCL</li>
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<li> NaH<sub>2</sub>PO<sub>4</sub>.2H<sub>2</sub>O (11.3 g/L)</li>
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<h4> Stock solutions </h4>
 
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<li>NaNO3 (150.0 g/L)</li>
 
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<li>Trace metals (mixed to 750 mL to dissolve, and then brought up to 1 L):  CuSO4.5H2O (19.6 mg/L), ZnSO4.7H2O (44.0 mg/L), CoCl2.6H2O (22.0 mg/L), MnCl2.4H2O (360.0 mg/L), Na2MoO4.2H2O (12.6 mg/L) </li>
 
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<li> Na2SiO3.5H2O (22.7 g/L) </li>
 
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<li>Fe citrate (both constituents added to the same 1 L: ferric citrate (9.0g/L), Citric acid (9.0g/L)</li>
 
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<li>Vitamins:  create primary stocks of Biotin (10.0 mg/100 mL) and vitamin B12 (10.0 mg/100 mL).</li>
 
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<li>Create a working solution of 100 mL, fresh every three months, containing: 1.0mL primary stock Biotin, 1.0 mL Vitamin B12, 20.0 mg Thiamine HCL).</li>
 
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<li> NaH2PO4.2H2O (11.3 g/L)</li>
 
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<h4>Further purification (Plasmid from putida)</h4>
 
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<li>Layer the resuspended DNA on a 2.5mL bed of saturated CsCl in a polymer tube.  Centrifuge for 14hr at 14 000 rpm in a fixed-angle 30 rotor at 2°C.  After the run, ~25mL of liquid can be discarded from the top without disturbing the remainder.  Mix the lower part to form the concentrated lysate.</li>
 
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<li>Slowly dissolve ~5.7g CsCl to the concentrated lysate, until the refractive index is 1.399.  Mix solution with Syber-safe or gel-red.  Centrifuge for 40hr in a Spinco fixed-angle rotor 50 at 105 000 x g at 12°C.  After this run, 2 well-separated bands should be able to be seen.  Alternatively, do only this spin where DNA mixed with CsCl is concentrated to a refractive index of 1.399 with the fluorescent DNA stain.</li>
 
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<li>Because the DNA was infused with dye, 2 well-separated bands will appear.  The upper band is linear and non-circular DNA (junk).  The lower band is the plasmid of interest.  Remove the upper layer with a micropipette.  After it is removed, pool bands from several tubes centrifuge again SW50.1 rotor for 20h at 40 000rpm.  Again there will be 2 bands and the lower band is the desired intact plasmid.</li>
 
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Latest revision as of 04:05, 29 September 2011


Preparation of f2 Media for Algae

This protocol is used to prepare f2 media for algal growth.

Stock solutions

  • NaNO3 (150.0 g/L)
  • Trace metals (mixed to 750 mL to dissolve, and then brought up to 1 L): CuSO4.5H2O (19.6 mg/L), ZnSO4.7H2O (44.0 mg/L), CoCl2.6H2O (22.0 mg/L), MnCl2.4H2O (360.0 mg/L), Na2MoO4.2H2O (12.6 mg/L)
  • Na2SiO3.5H2O (22.7 g/L)
  • Fe citrate (both constituents added to the same 1 L: Ferric citrate (9.0g/L), Citric acid (9.0g/L)
  • Vitamin primary stocks: of Biotin (10.0 mg/100 mL) and vitamin B12 (10.0 mg/100 mL).
  • Vitamin working solutions (made fresh every three months) containing: 1.0mL primary stock Biotin, 1.0 mL Vitamin B12, 20.0 mg Thiamine HCL
  • NaH2PO4.2H2O (11.3 g/L)

    • Procedure

    1. Add 0.5 mL of each of stock solutions 1-5 (listed below) to 1 L of seawater
    2. Split into flasks and autoclave at 121 °C (15 PSI) for 15 minutes
    3. Prepare phosphate solution (see below) and autoclave dilute phosphate stock at 121°C (15PSI, 15 mins). After cooling, dispense aseptically with sterilised automatic dispenser. This component is autoclaved separately in order to prevent precipitation


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