Protocol for ssDNA/RNA Ligation (M0319)
- Set up the following reaction in a sterile microfuge tube:
- Incubate at 65°C for 1 hour. For DNA-DNA Ligations the ssDNA to 5’ AppDNA ligation occurs with reduced efficiency, therefore in these cases we recommend extending the incubation time overnight.
- We recommend using a thermocycler or heated top for reaction incubation to reduce evaporation.
- For inactivation of the enzyme we recommend Proteinase K treatment or heat-kill at 90°C for 3 minutes.
- Proteinase K treatment is recommended to release RNA bound by the ligase; or you may see a smear on a gel.
|ssDNA/RNA Substrate||20 pmol (1 pmol/ul)|
|5´ App DNA Oligonucleotide||200 pmol (10 pmol/µl)|
|10X NEBuffer 1||2 µl|
|50 mM MnCl2 (for ssDNA ligation only)||2 µl|
|Thermostable 5´ App DNA/RNA Ligase||2 µl (40 pmol)|
|Nuclease-free Water||to 20 µl|