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Molecular Cloning Protocols

Transforming Competent E.coli with the Plasmid:

We chose the biobrick K137076 to ligate to the pvdQ gene for the following reasons:

• Take out the competent E-coli cells from the -80 freezer. (Keep all tubes on ice).  

• Add 1uL of the plasmid DNA in 100uL competent cells (if from Kit). For transformation of ligated product, add 10uL of the ligated plasmid DNA in 100uL competent

  cells.

• Incubate on ice for 10 min.

• Place in water bath at 42°C for 90s.

• Place immediately back on ice for at least 2 min.

• Add 800uL LB broth. Incubate for 1hr at 37°C shaker.

• Centrifuge at 130rpm for 5min to remove supernatant. Re-suspend the pellet in about 10uL of supernatant.

• Spread the entire re-suspended pellet on ampicillin agar dishes.

• Incubate for 12-16 hours at 37 °C.

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.