Team:Waterloo/Notebook/Protocols/Transformation

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<b><u><big>Transformation</big></u></b><br>

<b>Before you start </b><br>
Cells lose their competence once thawed, so be sure to:<br>
a) keep them on ice at all times.<br>
b) use cells immediately after thawing.<br>
c) discard any unused thawed cells. <br>

<b>Materials </b><br>
Competent cells<br>
DNA<br>
Ice + Ice bucket  <br>
1.5mL microfuge tube<br>
Glass Spreader<br>
Ethanol <br>
Agar Plate with desired antibiotic resistance<br>
Alcohol burner <br>

<b>Instructions</b><br>
1.  Thaw competent cells on ice. Include 2 extra ones for a Negative (no DNA) and Positive control. <br>
2.  In a 1.5 mL microfuge tube, add 50 μL of cells and 1 μL of DNA, and pipette up and down to mix. <br>
3.  Incubate on ice for 30 min.<br>
4.  Transfer directly to 42C heat block for 45 s to “heat shock”.<br>
5.  Transfer to ice for 2 min.<br>
6.  Add 500 μL of LB. <br>
7.  Incubate at 37C for 1 h on the shaker. <br>
8.  Centrifuge tubes at 13,000rpm for 2 mins.<br>
9.  Decant most of the supernatant. Leave approximately 50 μL.<br>
10. Prepare and label plates with date and construct names. Let the plates dry in the flow hood if necessary. <br>

<i>For each sample, the following steps must be carried out quickly in the laminar flow hood to ensure proper spreading of cells.</i><br>
11. In the laminar flow hood, resuspend pellet in remaining liquid by pipetting up and down. <br>
12. Transfer to center of plate. <br>
13. Dip glass spreader in ethanol and flame. <br>
14. Let the glass spreader cool by gently touching the agar. <br>
15. Spread liquid around plate until it appears dry. <br>
16. Seal plates with Parafilm or put in a bag. <br>
17. Incubate inverted in 37C incubator overnight.<br>