Protocol for cleavage with Tth Endonuclease IV (NEB #M0294)
1) Reaction set-up:
| DNA | Up to 10 pmol |
| ThermoPol Reaction Buffer (10X) | 5 µl |
| Tth Endonuclease IV |
1 µl (10 units) |
| H2O | Up to 50 µl |
2) Incubate at 65°C for 1 hour.
Notes:
- * An AP site is created by treating 10 pmol of a 60-mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.
- Place reaction in Thermocycler or overlay with mineral oil to reduce evaporation.
- Enzyme stability above 80°C is assured by adding ZnCl2 to a final concentration of 25 μM in the reaction.
- Not recommended in NEBuffers 1-4 and Thermopol II Buffer.
