Methods in Molecular Biology • 16 Enzymes of Molecular Biology

(Nancy Kaufman) #1

Ligases 225



  1. Experimental Procedures
    5.1. Uses of RNA Ligase
    The first obvious use of RNA ligase is in synthesizing oligomers.
    Modification of preexisting RNA molecules is perhaps a more interesting
    application. This may involve merely the end-labeling of an RNA moiety
    (69, 70), or alternatively, bases can be removed by periodate oxidation and
    13-elimination, and then modified bases can be added by RNA ligase to
    examine the restored function (71). Internal modification can also be
    achieved after nicking or treatment with RNAse H (72). The enzyme can
    also be used to join DNAs (69), although this requires a large concentra-
    tion of enzyme and a longer time.


5.2. Storage and Stability
T4 RNA ligase is available at specific activities of 1000-4000 U/mg
protein corresponding to 1-4 U/ktL. The enzyme should be diluted to
the required concentration in storage buffer, which is similar to that
used for T4 DNA ligase. In this case, the Tris-HCl concentration may
be raised to 50 mM, and KC1 may be omitted. The enzyme should be
held at -20°C.


5.3. Reaction Conditions
As with all manipulations involving RNA, great care should be
taken to eliminate the presence of ribonucleases. In practice, it is usu-
ally sufficient to ensure that all solutions and vessels have been auto-
claved prior to use.
T4 RNA ligase reaction buffers commonly consists of:



  • 50 mM HEPES/NaOH pH 7.5 (or 50 mM Tris-HC1, pH 8.0)

  • 10 mM MgCI 2 (10-18 raM)

  • 20 mM DTT (3-33 mM)

  • 1 mM ATP (0.1-1.0 mM)

  • 10% (v/v) DMSO

  • 100 lag/mL BSA (10-100 lag/mL)


Common alternative concentrations are shown in parentheses.
The substrate termini should be present in a concentration of 1-100
~k/(corresponding to 0.5-20 lag RNA), in a reaction vol of 20-50/pL.
The amount of RNA ligase required will vary with each application
within the range 0.1-16.0 U.

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