Ligation of an oligo to single stranded DNA (cDNA) using T4 RNA Ligase 1(M0204)
Overview
Similar conditions are used for ligation anchored PCR (1).
Protocol
- Set up a 20 μl reaction as follows:
1 X Reaction Buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT)
25% (wt/vol) PEG 8000
1 mM hexamine cobalt chloride (optional)
1 μl (10 units) T4 RNA Ligase
1 mM ATP - Incubate at 25°C for 16 hours.
- Stop the reaction by adding 40 μl 10 mM Tris-HCl pH 8.0, 2.5 mM EDTA.
References
- Troutt, A.B., et al. Proc. Natl. Acad. Sci. USA. 89. 9823-9825. 1992.
