You will have 100 L of bacteria per transformation. Keep the bacteria on ice.

Pipette 20 L of your ligation reaction directly into the tube containing the competent bacteria and mix gently by flicking the tube with your finger.

Allow the DNA to bind the bacteria on ice for 40 minutes.

Heat shock the bacteria at 42 C for 45 seconds.

Question: Why do we heat shock the bacteria?

Let the bacteria cool on ice for 2 minutes.

Add 800 L of LB to the bacteria and mix by inversion.

Allow the bacteria to incubate at 37 C for 1 hour on a shaker.

Question: What is happening during this hour? Why did we not use LB-antibiotic media?

Calculate the volume of vector and PCR product that you need for each of the reactions. Setup a reaction mixture of 20L to perform the three ligation reactions: - Tube 1 = non-phosphatase treated PstI digested pUC19, without insert - Tube 2 - phosphatase treated PstI digested pUC19, without insert - Tube 3 = phosphatase treated PstI digested pUC19, with insert Question: What role does each of the three (3) ligation reactions play? (ex. control what type? to control what? sample?)