T3 RNA Polymerase Protocol

Product No. RPOLT3-RO

Protocol

Removal of DNA:

Template DNA can be removed by direct addition of 20 units DNase, RNase-free to the IVT reaction mix and incubation for 15 min at +37 °C. This may not always be sufficient due to:
Incorporation of nucleotides into RNA generates pyrophosphate which inhibits DNase. For this reason, it is best to precipitate the nucleic acids from the IVT reaction and to add DNase after redissolving the the nucleic. A brief description of what to do is:

  1. Stop the IVT reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or heat to +65 °C.
  2. Ethanol precipitate the nucleic acids.
  3. Redissolve in 10 mM Tris-HCl (pH 7.5), 1 mM MnCl2.
    Note: About the use of manganese, and not magnesium: In the presence of Mg2+, DNase I produces nicks in
    - duplex DNA, while in the presence of Mg2+, the enzyme produces
    - double-stranded breaks in the DNA

  4. Add 1 Unit DNase I (RNase free), and incubate up to 15 min at +37 °C.
  5. Perform phenol/chloroform extraction, and re-precipitate the nucleic acids.

Materials

     
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