Digest and Ligation

Goal

• Goal: To insert a fragment of DNA into a plasmid using restriction enzymes and inserts.

Pre-Protocol Questions

1. Are you familiar with the NEBioCalculator and NEB Ligation Protocol? If not, refer to the links below.

The protocols for digest and ligation are from New England Biolab's "NEB Cloner" tool: http://nebcloner.neb.com/#!/redigest. and NEBioCalculator:"https://nebiocalculator.neb.com/#!/ligation and NEB Ligation Protocol with T4 Ligase: https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202.

Restriction Enzymes in the MEE Lab (and Buffer to Use)

• Acc651 (3.1 Buffer)
• BamHI-HF (CutSmart Buffer)
• BglII (3.1 Buffer)
• BsaI-HF (CutSmart Buffer)
• BsrGI-HF (CutSmart Buffer)
• DraI (CutSmart Buffer)
• EcoRI-HF (CutSmart Buffer)
• EcoRV-HF (CutSmart Buffer)
• FspI (CutSmart Buffer)
• HindIII-HF (CutSmart Buffer)
• KpnI-HF (CutSmart Buffer)
• MluI-HF (CutSmart Buffer)
• NcoI-HF (CutSmart Buffer)
• NdeI (CutSmart Buffer)
• NheI-HF (CutSmart Buffer)
• PstI-HF (CutSmart Buffer)
• PvuI-HF (CutSmart Buffer)
• PvuII-HF (CutSmart Buffer)
• SacI-HF (CutSmart Buffer)
• SalI-HF (CutSmart Buffer)
• SnaBI (CutSmart Buffer)
• SphI-HF (CutSmart Buffer)
• SspI-HF (CutSmart Buffer)
• XhoI (CutSmart Buffer)

Digestion

1. Prior to digesting DNA, purify DNA amplified via PCR to remove primers and to change the buffer.

a. Do this with the Zymogen DNA Clean and Concentrator Kits above the microwave.

2. Beforehand, turn on 37 degrees water bath (this is for the incubation period) and dry bath at 80 degrees (if you are heat inactivating the restriction enzymes).

3. Answer these questions:

a. What volume of DNA you will need to add in order to digest 1 µg?

b. Are you digesting with multiple restriction enzymes that require different buffers? If so, please talk to Eric.

c. Are you stopping here overnight (or longer)? Then, after the digestion, heat inactivate the enzymes at 80 degrees for 20 minutes.

4.

For a 50 µl reaction:

DNA	1 µg
10X Buffer (CutSmart or 3.1, depending on the enzyme)	5 µl (1X)
Each enzyme	1.0 µl
Nuclease-free Water	to 50 µl

OR

For a 10 µl reaction:

DNA	150 ng
10X Buffer (CutSmart or 3.1, depending on the enzyme)	1 µl (1X)
Each enzyme	0.2 µl
Nuclease-free Water	to 10 µl

5. Incubate reactions in a 1.5mL microcentrifuge tube at 37 degrees for at least 1 hour.

6. If necessary, store this DNA in the -20 freezer or in the 4 degree refrigerator for later use.

7. Purify (with the DNA Clean and Concentrator Kit) both the insert and plasmid vector DNA separately to remove the restriction enzymes.

Ligation

Navigate to the NEBioCalculator: https://nebiocalculator.neb.com/#!/ligation .

1. Insert these three values into the calculator.

a. Length in bp:

b. Vector DNA length

c. Vector DNA mass: enter 50 ng.

2. Use the (3:1) insert: vector ratio values.

3. Based on the amount of ng DNA resulting from the calculation, determine the appropriate volume of DNA to add to the ligation.

a. Be sure to use the T4 DNA ligase (with 10x Ligase Buffer) protocol on NEB's website: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

4. Thaw and resuspend ligase buffer at room temperature prior to performing the reaction.

a. The exact volumes of vector and insert to add to the ligation reaction will depend on results from the NEBioCalculator.

1. Add 2 uL of 10X Ligase Buffer to a 0.6 mL tube.

2. Add the vector. How much? 50ng of DNA, so the volume will depend on the concentration of the vector.

3. Add three-times the molar amount of insert. This will be a variable amount; be sure to use the above calculator to find the volume needed.

4. Add 1 uL of T4 Ligase

5. Fill the remaining volume with molecular grade water (in 4 degree fridge) to 20 uL.

6. Gently mix the reaction by gently pipetting up and down.

7. Allow the reaction to run for 10 minutes at room temperature.

8. Heat inactivate at 65 degrees for 10 minutes.

9. Either proceed to transformation, or store DNA in -20 degrees.