Protocol ligation and transformation

This week, we will be performing a DpnI digest on our OE-PCR cloning reaction, then transforming it into chemically competent DH5α E. coli by heat shock, and plating them out on LB ampicillin plates.

DpnI Digest, Ligation, and Transformation of Chemically Competent E. coli

1. Add 1 L of DpnI enzyme to your PCR reaction from last week.μ
2. Incubate in the PCR machine using the heat block incubation function, at 37°C for 15 minutes.
3. Ask a demonstrator for an aliquot DH5α chemically competent cells (they are being stored in the freezer). Allow the cells to thaw on ice for 5 minutes, then add 1 L of your reaction to your aliquot of cells. Incubate μ on ice for 5 minutes.
4. Heat shock the cells at 42°C for 30 seconds, and immediately return to ice. Incubate on ice for at least 2 minutes.
5. Add 50 L of SOC media, mix gently by flicking the tube, then incubate μ the cells at 37°C in a water bath for 30 minutes. While waiting, prepare your plate.

Prepare your Plates

Spread 40 L of 1M IPTG, and 40 L of 40mg/mL evenly on the ampicillin μ μ plate. You need to make sure the entire plate is evenly covered.

Plating

Always label your plates with your names, the date, and MBHG. When writing on a plate, always write on the back of the plate, not on the lid. The lid can come off and get lost, the back can’t.

Pipette 25 L of the transformed cells onto the plate and spread in a fanμ shape from the line to the plate edges. When you are finished, seal your plate with Parafilm by stretching the Parafilm around the edge of the closed plate (if you don’t know how to do this, ask a demonstrator), and turn in to the instructor for incubation.