Team:Calgary/Notebook/Protocols/Process5

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TITLE=Plasmid Extraction|
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TITLE=Preparation of f2 Media for Algae|
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<p>
<p>
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This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.</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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<br>
<br>
<h4> Procedure </h4>
<h4> Procedure </h4>
<ol>
<ol>
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<li>Mix together</li>
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<li>Add 0.5 mL of each of stock solutions 1-5 (listed below) to 1 L of seawater</li>
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<li>10g tryptone</li>
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<li>Split into flasks and autoclave at 121 &deg;C (15 PSI) for 15 minutes </li>
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<li>5g yeast extract</li>
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<li>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</li>
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<li>5g NaCl</li>
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<li>20% sucrose (autoclaved)</li>
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<li>Triton X-100</li>
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<li>500mM EDTA stock (pH 8.0)</li>
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<li>Tris-HCl 50mM</li>
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<li>NaCl 3M</li>
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<li>Isopropanol</li>
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<li>Autoclaved Milli-Q water</li>
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</ol>
</ol>
<br>
<br>
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<h4> Procedure </h4>
 
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<ol>
 
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<li>Inoculate a loopful of Pseudomonas sp. at 25oC, in 10mL LB broth (10g tryptone; 5g yeast extract, 5g NaCl, 1000mL distilled H2O, pH 7.0), and incubate for 16-18hr.</li>
 
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<li>Centrifuge 1.5 ml of a 16-18hr bacterial culture for 1 min at 11,500 x g in a polypropylene centrifuge tube.</li>
 
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<li>Remove supernatant by aspirating, leaving the pellet as dry as possible.</li>
 
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<li>Add to each tube, 400µL of 8% sucrose, 5.0% Triton X-100, 50 mM EDTA, and 10mM Tris HCI (pH 8.0). Mix well.</li>
 
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<li>Add 50µL of freshly prepared lysozyme solution (10mg/mL in 10mM Tris HCl, pH8), mix by inverting 3X.  Lysozyme digests bacterial cell wall.</li>
 
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<li>Immediately incubate at 100oC for 10, 20, 40, 80s.</li>
 
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<li>Spin for 10min 11,500Xg at room temp.</li>
 
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<li>Remove pellet with sterile forceps.</li>
 
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<li>Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.</li>
 
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<li>Incubate 30min at -20oC.</li>
 
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<li>Centrifuge 15min for 11,500Xg at 4oC.</li>
 
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<li>Decant supernatant, invert and drain on clean paper towel.</li>
 
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<li>Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).</li>
 
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<li>Incubate for 1hr at 4oC in dark.  </li>
 
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<li>Run a small amount of this sample on gel electrophoresis on 0.7% (w/v0) agarose.  With the rest, submit to further purification.</li>
 
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</ol>
 
<br>
<br>
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<h4>Further purification (Plasmid from putida)</h4>
 
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<ol>
 
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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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