DNA Reaction - Ligation

Jeff Lawrence

1. Phosphatase the vector ends. Leave the insert ends with 5' phosphates. Kinase PCR products if necessary.

2. Use a 2:1 ratio of insert ends to vector ends. use 5:1 ratio is blunt ends are used.

3. Final DNA concentration should be 200 fmol/10 mL, or 0.02 µM. For blunt ended ligation, final DNA concentration should be 0.2 µM

	1 µL	Vector, about 17 fmol
	2 µL	Insert, about 85 fmol
	1 µL	5X Ligation Buffer.
	1 µL	T4 DNA Ligase

4. Incubate at 16° for at least 6 hours. Heat reaction to 70° for 3 min to inactivate enzyme.

5. Add 15 µL 95% Ethanol. Incubate at -20° for 2 hrs.

6. Centrifuge for 15 min at 4°. Remove supernatant.

7. Add 400 µL 70% Ethanol. Mix well to dislodge pellet. Microfuge at 4° for 2 min. Discard supernatant. Repeat for a second rinse if DNA is to be used in electroporation.

8. Resuspend in 10 µL ddH₂O.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu