Notes:

1. Colonies grown on plate were selected for colony PCR screening.

2. Correct colonies were then cultured in 5 mL LB Broth with 0.5uL ampicillin for subsequent screening or mass culturing.

3. Mass culturing involved adding 200uL of the culture into 35mL LB Broth with 35uL ampicillin.

Miniprep to Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit):

• Transfer some of the 5mL bacterial culture into a microcentrifuge tube. Pellet by centrifugation at 13,500 rpm for 1 min. Repeat till all the culture has been pelleted.

• Resuspend the pellet in 250uL Buffer P1.

• Add 250uL Buffer P2 (Lysis Buffer). Mix thoroughly by inverting the tube 4-6 times. Do not allow prolonged lysis.

• Immediately add 350uL Buffer N3 (Neutralization Buffer). Mix immediately by inverting the tube.

• Centrifuge for 10 minutes at 13,500 rpm.

• Apply the resulting supernatant to the QIAprep spin column by pipetting. Centrifuge for 1 minute at 13,500 rpm.

• Wash the QIAprep spin column by applying 0.75mL Buffer PE. Centrifuge for 1 minute at 13,500 rpm.

• Centrifuge for an additional 1 minute to remove residual ethanol.

• Place the QIAprep spin column in microcentrifuge tube. Elute DNA by adding 50uL warm H₂O.

• Centrifuge for 1 minute at 13,500 rpm.