Team:UC Davis/Protocols

From 2013.igem.org

(Difference between revisions)
Line 192: Line 192:
<li>10 g      NaCl</li>
<li>10 g      NaCl</li>
<li>5 g        Yeast Extract</li>
<li>5 g        Yeast Extract</li>
-
1 L  Total
+
1 L  Total (add dH<sub>2</sub>0 to reach total volume)
<li>Add 15 g Agar, if being poured into plates.</li>
<li>Add 15 g Agar, if being poured into plates.</li>
<li>Autoclave, when cool add antibiotics if desired.</li>
<li>Autoclave, when cool add antibiotics if desired.</li>
Line 229: Line 229:
<li>Preheat water bath to 42º C.</li>
<li>Preheat water bath to 42º C.</li>
<li>Thaw competent cells on ice for 10 minutes.</li>
<li>Thaw competent cells on ice for 10 minutes.</li>
-
<li>Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes. </li>
+
<li>Use 50 µL chemically competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (e.g. PUC19). Swirl gently to mix, store on ice 5 minutes. </li>
<li>Heat shock in 42º C water bath for 45 seconds.</li>
<li>Heat shock in 42º C water bath for 45 seconds.</li>
-
<li>Cool cells in ice bath for a few minutes.</li>
+
<li>Cool cells in ice bath for 1-2 minutes.</li>
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li>
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li>
-
<li>Transfer 200 µL of culture per plate (containing the appropriate antibiotic).</li>
+
<li>Transfer 200 µL of culture to a plate (containing the appropriate antibiotic).</li>
<li>Spread using glass beads.</li>
<li>Spread using glass beads.</li>
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.</li>
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.</li>

Revision as of 23:35, 18 October 2013

Protocols