Digest and Ligation

The protocols for digest and ligation are from New England Biolab's "NEB Cloner" tool: http://nebcloner.neb.com/#!/redigest.

Digestion: (1.5hrs)

Prior to digesting DNA, be sure to purify the DNA amplified with Q5 polymerase to remove excess primers. Do this with the Zymogen DNA Clean and Concentrator Kits above the microwave.

Beforehand, turn on 37 degrees water bath and 65 degrees (for ligation heat inactivation). Calculate how much DNA you will need to add in order to digest 1ug. You can store this DNA in the -20 freezer for later use.

1. Go to the cloner tool link: http://nebcloner.neb.com/#!/redigest.
2. Simply select your digestion enzymes from dropdown menu for either a single or double restriction enzyme digestion. The buffer efficiency for each of the enzymes will be listed for the available NEB buffer options.
   1. Be aware of star* activity next to listed efficiency, indicating that the buffer may alter specificity of restriction enzymes. See baseball card binder for more information on star activity between pairs of restriction enzymes and their most efficient buffers.
3. Click on "Detailed Protocol" at the top middle of the page to see reagents and corresponding volumes to add to each reaction.

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

Between the digestion and ligation steps, purify (with the DNA Clean and Concentrator Kit) both the insert and plasmid vector DNA to remove restriction enzymes.

Ligation: (0.5hrs)

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

1. There are three values to insert into the calculator.
   1. Insert length in bp:
   2. Vector DNA length
   3. Vector DNA mass: enter 50ng.

Go with the (3:1) insert:vector ratio values. Based on the amount of ng DNA resulting from the calculation, determine the appropriate volume of DNA to add to the ligation. 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

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

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

1. Add 2uL of 10X Ligase Buffer to a 0.6mL tube.
2. Add XX amount of insert
3. Add XX amount of vector
4. Add 1uL of T4 Ligase
5. Fill remaining volume with molecular grade water (in 4 degree fridge) to 20uL.
6. Gently mix the reaction by gently pipetting up and down.
7. Allow 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.