Team:Calgary/Notebook/ProtocolManual/General

From 2014.igem.org

(Difference between revisions)
Line 9: Line 9:
<li>Add TAE Buffer (100 mL) and Agarose (1g) to a flask [for a 1% gel]</li>
<li>Add TAE Buffer (100 mL) and Agarose (1g) to a flask [for a 1% gel]</li>
<li>Cover with plastic wrap, poke hole in top. Then microwave flask until agarose is fully dissolved; avoid boiling</li>
<li>Cover with plastic wrap, poke hole in top. Then microwave flask until agarose is fully dissolved; avoid boiling</li>
-
<li>Take flask to fume hood, and allow to cool to touch. Add SYBR safe (4 μL) to agarose, gently swirl to mix</li>
+
<li>Take flask to fume hood, and allow to cool to touch. Add REDsafe (4 μL) to agarose, gently swirl to mix</li>
<li>Gently pour agarose into assembled gel casting tray, removing any bubbles with a pipette tip</li>
<li>Gently pour agarose into assembled gel casting tray, removing any bubbles with a pipette tip</li>
<li>Allow gel to solidify until translucent. Then transfer to running apparatus filled with TAE buffer</li>
<li>Allow gel to solidify until translucent. Then transfer to running apparatus filled with TAE buffer</li>
<li>Load samples containing 3 μL loading dye and ~10-15 μL of DNA</li>
<li>Load samples containing 3 μL loading dye and ~10-15 μL of DNA</li>
<li>Run gel at 110V until the dye is ~2/3 of the way down the gel (approx. 40 mins)</li>
<li>Run gel at 110V until the dye is ~2/3 of the way down the gel (approx. 40 mins)</li>
 +
</p>
 +
<p>
 +
<b>Preparing Chemically Competent <i>E. coli</i> Cells</b>
 +
<ol type=1>
 +
<li>Inoculate 5-10mL LB with Top10 E. coli culture at 37°C shaking over night</li>
 +
<li>Subculture 1mL of bacteria into 50mL LB at 37°C shaking until OD<sub>600</sub> is 0.4-0.6 (~2.5 hr)</li>
 +
<li>Centrifuge the subculture at max for 20 min at 4°C</li>
 +
<li>Resuspend pellet in 12.5mL cold CaCl<sub>2</sub> (50mM, 15% glycerol)</li>
 +
</ol>
</p>
</p>
 +
 +
</section>
</section>
</html>
</html>

Revision as of 22:17, 14 October 2014

General Protocols

Agarose Gel Electrophoresis

  1. Add TAE Buffer (100 mL) and Agarose (1g) to a flask [for a 1% gel]
  2. Cover with plastic wrap, poke hole in top. Then microwave flask until agarose is fully dissolved; avoid boiling
  3. Take flask to fume hood, and allow to cool to touch. Add REDsafe (4 μL) to agarose, gently swirl to mix
  4. Gently pour agarose into assembled gel casting tray, removing any bubbles with a pipette tip
  5. Allow gel to solidify until translucent. Then transfer to running apparatus filled with TAE buffer
  6. Load samples containing 3 μL loading dye and ~10-15 μL of DNA
  7. Run gel at 110V until the dye is ~2/3 of the way down the gel (approx. 40 mins)

    8. Preparing Chemically Competent E. coli Cells

    1. Inoculate 5-10mL LB with Top10 E. coli culture at 37°C shaking over night
    2. Subculture 1mL of bacteria into 50mL LB at 37°C shaking until OD600 is 0.4-0.6 (~2.5 hr)
    3. Centrifuge the subculture at max for 20 min at 4°C
    4. Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)

Retrieved from "http://2014.igem.org/Team:Calgary/Notebook/ProtocolManual/General"