Team:Calgary/Notebook/Protocols/Process5

From 2011.igem.org

(Difference between revisions)
Line 22: Line 22:
<li>NaNO3 (150.0 g/L)</li>
<li>NaNO3 (150.0 g/L)</li>
<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>  
<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>  
-
<li>Remove supernatant by aspirating, leaving the pellet as dry as possible.</li>
+
<li> Na2SiO3.5H2O (22.7 g/L) </li>
-
<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>
+
<li>Fe citrate (both constituents added to the same 1 L: ferric citrate (9.0g/L), Citric acid (9.0g/L)</li>
-
<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>
+
<li>Vitamins .</li>
-
<li>Immediately incubate at 100oC for 10, 20, 40, 80s.</li>
+
<li> NaH2PO4.2H2O (11.3 g/L)</li>
-
<li>Spin for 10min 11,500Xg at room temp.</li>
+
 
-
<li>Remove pellet with sterile forceps.</li>
+
-
<li>Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.</li>
+
-
<li>Incubate 30min at -20oC.</li>
+
-
<li>Centrifuge 15min for 11,500Xg at 4oC.</li>
+
-
<li>Decant supernatant, invert and drain on clean paper towel.</li>
+
-
<li>Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).</li>
+
-
<li>Incubate for 1hr at 4oC in dark.  </li>
+
-
<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>
+
</ol>
</ol>

Revision as of 01:04, 27 September 2011


Preparation of f2 media for algae

This protocol is used to prepare f2media for algae growth


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

Stock solutions

  1. NaNO3 (150.0 g/L)
  2. 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)
  3. Na2SiO3.5H2O (22.7 g/L)
  4. Fe citrate (both constituents added to the same 1 L: ferric citrate (9.0g/L), Citric acid (9.0g/L)
  5. Vitamins .
  6. NaH2PO4.2H2O (11.3 g/L)

Further purification (Plasmid from putida)

  1. 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.
  2. 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.
  3. 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.

Retrieved from "http://2011.igem.org/Team:Calgary/Notebook/Protocols/Process5"