Ligations
Set-up:
1. Run a gel of your digestions and take a picture.
2. Estimate the intensities of your insert and vector where your insert (usually less intense) is considered 1 and your vector is x times as bright.
3. Once intensity is determined perform the following equation:
L = Ligation volume
S = Size of fragments
I = Intensity of fragments
V = Volume ran down gel
i = Insert
v = Vector
4. Ligation volume (Lv) for the vector should be approximately 2 µl.
5. The ratio for the ligation in the previous equation is 3:1 insert:vector but can be done 6:1 by replacing the 3 with a 6.
Reaction Mix:
- 2 µl – Vector
- Li µl – Insert
- 2 µl – 10x Buffer
- dH20 to 19 µl
- 1 µl – T4 DNA Ligase
- TOTAL – 20 µl
Procedure:
1. Add appropriate amounts of dH20, Vector, and Insert in a PCR tube (200 µl). Once added, heat mixture to 65o C for 5 min. This will remove all items that are already annealed to each other causing inefficient ligation.
2. Add then appropriate amounts of 10x Buffer and T4 DNA Ligase. Make sure not to pipette up and down or vortex to mix, this will shear the ligase. Gently swirl with the tip of your pipette to mix.
3. Incubate reaction at room temperature for 30 minutes.
4. After incubation heat inactivate the ligase by another incubation at 80o C for 20 min.
NOTE: The ligase buffer should smell like wet dog. If you smell nothing from the tube, then the buffer is old and useless.
Can also use the Ginkgo Bioworks protocol:
1. Get the 10X T4 DNA ligase buffer and the T4 DNA ligase from the 20 degree storage.
2. Put them on ice to thaw.
3. In a PCR tube add 11 uL of water.
4. Add 2 uL of each digest to the tube.
5. Use a pipet to thoroughly mix the T4 DNA ligase buffer and resuspend the precipitate in the bottom. Then add 2 uL to the PCR tube.
6. Add 1 uL of T4 DNA ligase.
7. Flick the tube to mix it. If necessary, put it in the microcentrifuge to get all the liquid back to the bottom.
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