DNA Template Preparation (E2040)

Protocol

1. Plasmid Templates
   
   Completely linearized plasmid template of highest purity is critical for successful use of the HiScribe T7 High Yield RNA Synthesis Kit. Quality of the template DNA affects transcription yield and the integrity of RNA synthesized. The highest transcription yield is achieved with the highest purity template. Plasmid purified by many laboratory methods can be successfully used, provided it contains mostly supercoiled form, and is free from contaminating RNase, protein, RNA and salts.
   
   To produce RNA transcript of a defined length, plasmid DNA must be completely linearized with a restriction enzyme downstream of the insert to be transcribed. Circular plasmid templates will generate long heterogeneous RNA transcripts in higher quantities because of high processivity of T7 RNA polymerase. NEB has a large selection of restriction enzymes; we recommend selecting restriction enzymes that generate blunt ends or 5´-overhangs.
   
   After linearization, we recommend purifying the template DNA by phenol/chloroform extraction:
   1. Extract DNA with an equal volume of 1:1 phenol/chloroform mixture, repeat if necessary.
   2. Extract twice with an equal volume of chloroform to remove residual phenol.
   3. Precipitate the DNA by adding 1/10th volume of 3 M sodium acetate, pH 5.2, and two volumes of ethanol. Incubate at -20°C for at least 30 minutes.
   4. Pellet the DNA in a microcentrifuge for 15 minutes at top speed. Carefully remove the supernatant.
   5. Rinse the pellet by adding 500 μl of 70% ethanol and centrifuging for 15 minutes at top speed. Carefully remove the supernatant.
   6. Air dry the pellet and resuspend it in nuclease-free water at a concentration of 0.5-1 μg/μl.
2. PCR Templates
   
   PCR products containing T7 RNA Polymerase promoter in the correct orientation can be transcribed. Though PCR mixture can be used directly, better yields will be obtained with purified PCR products. PCR products can be purified according to the protocol for plasmid restriction digests above, or by using commercially available spin columns (we recommend Monarch PCR & DNA Cleanup Kit, NEB #T1030). PCR products should be examined on an agarose gel to estimate concentration and to confirm amplicon size prior to its use as a template in the HiScribe T7 High Yield RNA Synthesis Kit. Depending on the PCR products, 0.1–0.5 μg of PCR fragments can be used in a 20 μl in vitro transcription reaction.
   
   
3. Synthetic DNA Oligonucleotides
   
   Synthetic DNA Oligonucleotides which are either entirely double-stranded or mostly single-stranded with a double-stranded T7 promoter sequence can be used in the HiScribe T7 High Yield RNA Synthesis Kit. In general, the yields are relatively low and also variable depending upon the sequence, purity and preparation of the synthetic oligonucleotides.