Ligation of an oligo to single stranded DNA (cDNA) using T4 RNA Ligase 1

Overview

Similar conditions are used for ligation anchored PCR (1).

Protocol

1. 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
2. Incubate at 25°C for 16 hours.
3. Stop the reaction by adding 40 μl 10 mM Tris-HCl pH 8.0, 2.5 mM EDTA.

References

1. Troutt, A.B., et al. Proc. Natl. Acad. Sci. USA. 89. 9823-9825. 1992.