DNA Ligation Using T4 DNA Ligase and Bacterial Transformation

*We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector. The following ligation reaction of a 3.0kb vector and 0.5kb insert DNA uses the 1:3 vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA.

**Blue/white screening can be used with a variety of vectors. To use blue/white color screening for recombinants, plate the transformed cells on LB plates containing the appropriate antibiotic, 0.5mM IPTG and 40µg/ml X-gal. Incubate overnight at 37°C. An alternative to preparing plates containing X-gal and IPTG is to spread 20µl of 50mg/ml X-Gal and 100µl of 0.1M IPTG onto LB ampicillin plates and allow these components to absorb for 30 minutes at 37°C prior to plating cells. HB101 and BL21(DE3)pLysS competent cells cannot be used for blue/white color screening.

Adjust the pH to 7.5 with NaOH. Add 15g of agar per liter. Autoclave to sterilize. Allow the autoclaved medium to cool to 55°C. For LB ampicillin plates, add ampicillin to a final concentration of 100µg/ml. For LB tetracycline plates, add tetracycline to a final concentration of 10µg/ml. Store the plates at 4°C.

Plates containing tetracycline must be stored protected from light to maintain potency.

Add distilled water to 100ml. Filter sterilize.

Add Bacto®-tryptone, Bacto®-yeast extract, NaCl and KCl to 97ml of distilled water. Stir to dissolve. Autoclave and cool to room temperature. Add 2M Mg2+ stock and 2M glucose, each to a final concentration of 20mM. Bring the volume to 100ml with sterile, distilled water. The final pH should be 7.0.

Adjust the pH to 7.5 with NaOH. Autoclave to sterilize.

Assemble the following reaction in a sterile microcentrifuge tube:

We recommend performing a negative control ligation with no insert. Carry this reaction through transformation and plating. This allows you to determine how many background colonies you have on your plate. For a positive control reaction, simply perform a 15- to 30-minute ligation reaction under normal conditions using 1µg of a DNA digest marker (e.g., Lambda DNA HindIII Markers). Run the ligation reaction on a gel, and compare the size to the standard marker. You should see DNA of much higher molecular weight on the gel in the ligated marker. Another quick test is to cut a plasmid with a single restriction enzyme. Add this linearized vector to a ligation reaction and transform.

Incubate the reaction:

22–25°C for 3 hours for cohesive ends.

4°C overnight for cohesive ends.

15°C for 4–18 hours for blunt ends.

Chill sterile 17 × 100mm polypropylene culture tubes on ice, one per transformation. Use of a standard microcentrifuge tube reduces the transformation efficiency by approximately 50% due to inefficient heat-shock treatment of the cells.

Note: To determine transformation efficiency, we recommend assembling a separate transformation with 1µl (0.1ng) of circular plasmid DNA.

Remove frozen competent cells from –70°C, and place on ice for 5 minutes or until just thawed. Once the cells have thawed, pipet quickly or use chilled (4°C) pipette tips to prevent the cells from warming above 4°C.

Gently mix the thawed competent cells by flicking the tube, and transfer 100µl to each of the chilled culture tubes.

Add 1–50ng of DNA (in a volume not greater than 10µl) per 100µl of competent cells. Move the pipette tip through the cells while dispensing. Quickly flick the tube several times.

Immediately return the tubes to ice for 10 minutes.

Heat-shock the cells for 45–50 seconds in a water bath at exactly 42°C. Do not shake.

Immediately place the tubes on ice for 2 minutes.

Add 900µl of cold (4°C) SOC medium to each transformation reaction.

Incubate at 37°C with shaking (approximately 225rpm) for 60 minutes.

For each transformation reaction, we recommend diluting the cells 1:10 and 1:100 and plating 100µl of the undiluted, 1:10 and 1:100 dilutions on antibiotic plates (see Notes 1 and 2).

Incubate the plates at 37°C for 12–14 hours.

1. For transformations using a circular plasmid control DNA, we recommend diluting the cells 1:10, then plating 100µl on LB/antibiotic plates.
2. If desired, pellet the cells by centrifugation at 1,000 × g for 10 minutes, then resuspend in 200µl of SOC or LB medium and plate.

For more information, visit the Promega Subcloning Notebook at: http://www.promega.com/guides/subcloning_guide/