Team:Grinnell/Notebook/Protocols

From 2011.igem.org

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<li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).</li>
<li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).</li>
<li>Chill cells on ice for 15-120min.</li>
<li>Chill cells on ice for 15-120min.</li>
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<li>Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4 degrees C. Discard supernatant.</li>
+
<li>Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4&deg; C. Discard supernatant.</li>
<li>Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.</li>
<li>Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.</li>
<li>Harvest cells by cetrifugation. Discard supernatant.</li>
<li>Harvest cells by cetrifugation. Discard supernatant.</li>
-
<li>Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.</li>
+
<li>Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80&deg; C.</li>
</ol>
</ol>
<h3><a name="Transformation"></a>Plasmid Transformation</h3>
<h3><a name="Transformation"></a>Plasmid Transformation</h3>
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<li>Add 10uL DNA to cells.</li>
<li>Add 10uL DNA to cells.</li>
<li>Incubate tubes on ice for 30min.</li>
<li>Incubate tubes on ice for 30min.</li>
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<li>Incubate tubes at 42 degrees C for 90sec.</li>
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<li>Incubate tubes at 42&deg; C for 90sec.</li>
<li>Incubate tubes on ice for 2min.</li>
<li>Incubate tubes on ice for 2min.</li>
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<li>Add 300mL LB to cells and incubate shaking at 37 degrees C for 1hr.</li>
+
<li>Add 300mL LB to cells and incubate shaking at 37&deg; C for 1hr.</li>
<li>Spread cells on selective media</li>
<li>Spread cells on selective media</li>
-
<li>Incubate plates overnight at 37 degrees C.</li>
+
<li>Incubate plates overnight at 37&deg; C.</li>
</ol>
</ol>
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Revision as of 00:18, 9 June 2011

Grinnell Menubar

Competent Cells

  1. Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).
  2. Chill cells on ice for 15-120min.
  3. Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.
  4. Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
  5. Harvest cells by cetrifugation. Discard supernatant.
  6. Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80° C.

Plasmid Transformation

  1. Thaw 100uL aliquots of competent cells on ice.
  2. Add 10uL DNA to cells.
  3. Incubate tubes on ice for 30min.
  4. Incubate tubes at 42° C for 90sec.
  5. Incubate tubes on ice for 2min.
  6. Add 300mL LB to cells and incubate shaking at 37° C for 1hr.
  7. Spread cells on selective media
  8. Incubate plates overnight at 37° C.

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