Example 3
The in vitro transcription reactions can generate polynucleotides containing uniformly modified polynucleotides. Such uniformly modified polynucleotides can comprise a region or part of the polynucleotides of the invention. The input nucleotide triphosphate (NTP) mix can be made using natural and un-natural NTPs.
A typical in vitro transcription reaction can include the following:
-
- 1 Template cDNA—1.0
- 2 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, mM Spermidine)—2.0
- 3 Custom NTPs (25 mM each)—7.2 μl
- 4 RNase Inhibitor—20 U
- 5 T7 RNA polymerase—3000 U
- 6 dH2O—Up to 20.0 μl. and
- 7 Incubation at 37° C. for 3 hr-5 hrs.
The crude IVT mix can be stored at 4° C. overnight for cleanup the next day. 1 U of RNase-free DNase can then be used to digest the original template. After 15 minutes of incubation at 37° C., the mRNA can be purified using Ambion's MEGACLEAR™ Kit (Austin, TX) following the manufacturer's instructions. This kit can purify up to 500 μg of RNA. Following the cleanup, the RNA can be quantified using the NanoDrop and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred.
