We will follow the suggested protocol provided by Promega's 2X Rapid Ligation protocol. In order to do this, you will need to calculate the amount of insert you need with this formula:
X ng of insert = (3) (bp insert) (50 ng linearized plasmid-) ÷ (size of plasmid in bp)
Thaw the frozen 2X buffer at room temperature. After
the first thawing, it is best if you aliquot the 2X buffer in smaller volumes
to avoid repeated freeze/thaw cycles. If a precipitate is
present, vortex the solid DTT until it is back in solution. This usually
takes 1 - 2 minutes.
Set up the following 10 µL ligation reaction (If your DNA is too dilute,
you can scale up to 20 µL ligation volume and transform all
20 µL. Alternatively, you can add DNA together, speedvac to dryness and resuspend the DNA in 4 µL water.)
| ^ Digested Vector (50 ng) | 1 µL |
| # Insert (3:1 molar ratio insert:vector) | (< 3) x µL |
| Sterile water | 3µL - x µL |
| 2X ligation buffer | 5 µL |
| * 3 units T4 DNA ligase (keep cold) | 1 µL |
| Total Volume | 10.0 µL |
^ To conserve DNA, you can reduce the amount of plasmid to 10 ng. You can also change the ratio from 3:1 to 2:1 in the formula above.
# The 3 in the numerator takes into account the fact that you want to have
a 3:1 ratio of insert to vector. A standard ligation reaction uses 50 ng of plasmid for a
ligation.
* To conserve ligase, you can reduce the amount use from 1.0 µL to 0.5 µL T4 DNA ligase. If you do this, you will need 0.5µL water to bring the final volume to 10.0 µL.
Incubate for 5 minutes at room temperature. From this point, you may either freeze the ligation or go directly to transformation.
If your DNA is too dilute to fit into a 10µL reaction, you can combine the DNA in your ligation tube and speedvac it to dryness and then rehydrate with 4 µL water.
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