Shlo/notebook/general protocols

Ligation & Transformation: Courtesy of Perry

Ligation

Follow the protocol outlined in Roche Rapid DNA Ligation kit.

You need insert DNA and vector DNA, both compatibly digested. You want more insert than vector, about a 3:1 molar ratio. You can either nanodrop each sample for DNA concentration, and do the math, or you can do a simple 7ul:3ul mixture. Then add 10ul ligation buffer and mix. Finally add 1ul Rapid DNA ligase and mix. Let sit on the bench for 5 minutes.

You can either freeze the ligation mixture, or you can do it while your competent cells are thawing for transformation.

Transformation

Get Top10 chemically competent cells from the -80dC freezer, and let thaw on ice.

After the cells have thawed, divide and aliquot into as many tubes as transformations you're performing. There is 60ul in one tube; you can do as many as 6 transformations from one tube, 10ul competent cells each. Competent cells are expensive, and they lose competency when frozen/thawed a second time, so try to be economical.

Add the DNA plasmid (from ligation, Topo cloning, or plasmid purification).

Let the tubes sit on ice for 20 minutes. (Make sure the heat-block has water in the wells and is set to 42dC.)

Heat shock the tubes at 42dC for 30 seconds.

Cool the tubes on ice for 2 minutes.

Add 200ul SOC media.

Incubate the tubes in the incubator/shaker at 37dC for 1h. While they're incubating, label agar plates of the appropriate antibiotic, and pre-warm in the 37dC incubator cabinet.

Under a flame, pipette the mixture onto the agar plate, and spread with a sterile plastic spreader.

Leave the agar plates agar-side-up on the shelf in the 37dC incubator, and leave for 12-16 hours.