Circularization of R334C

Phosphorylation

1. Obtain two 1.5 mL tubes and label them with the DNA names.

First tube: R334C 1 (10ng/uL)

Second tube: R334C 2 (25ng/uL)

2. For each tube, write CIRC below the DNA name add the reagents in the following order:

5uL MBG water (Fridge A)

2uL linear PCR product (PCR Rack, Freezer A)

1uL T4 DNA ligase buffer (Enzyme Rack, Freezer A)

1uL 10mM ATP (Enzyme Rack, Freezer A)

1uL T4 PNK (Enzyme Rack, Freezer A)

3. Centrifuge for 5-10 seconds to ensure all solution is at the bottom of the tubes.

4. Incubate at 37C for 20 minutes . Write PHOS on the side of the tube.