Team:Wisconsin-Madison/Transformation of Plasmids into Competent Cells

Transformation of Plasmids into Competent Cells

1. Clean cuvettes with 95% Ethanol and UV in hood for about a minute, set on ice

2. Let competent cells and plasmids thaw on ice

3. Mix in cuvette:

• 40 μL of competent cells
• 1/3 – 1 μL plasmid

4. Let sit on ice for 5 min

5. Set Electroporator to ECORI and MS, align cuvette correctly, and shock

• Should get milli second shock between 4 - 6 ms
• ARC – too high of concentration of electrolights (plasmids)

6. Immediately after shock, add 950 μL of LB and mix well

7. Transfer to centrifuge tube

8. Let grow in water bath at 37 C (expressing antibiotic proteins)

• Amp – 40 minutes
• Cm – 1 hour
• Kam - 1 hour

9. Plate 50 μL of cells, make 100x and 10,000x dilutions (3 plates)

• Circle in center of plate, do not get sample in edges