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  • 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.