Experiment Protocols

In Vitro Transcription Troubleshooting

In vitro transcription describes the template-directed synthesis of RNA molecules; from short oligonucleotides to those of several kilobases. Such synthetic RNA transcripts are an important tool in structural, biochemical and genetic studies. Mistakes can lead to failed transcription or incorrectly-sized transcripts.

Below we list some of the common problems you may encounter, their cause, and how to solve them:

Problem: Failed Transcription

Possible Cause: Impure RNA template.
Solution:  Contaminants like salts can inhibit the activity of the RNA polymerase. Use a clean-up kit to desalt your template DNA.

Possible Cause: Incorrect linearized template.
Solution:  Verify that the sequence and restriction map are correct. Check an aliquot of purified DNA on an agarose gel.

Possible Cause: RNA degradation due to RNase contamination during plasmid purification.
Solution:  Use an RNase inhibitor.

Possible Cause: Inactive RNA polymerase.
Solution:  Always use a positive control template to ensure your in vitro transcription reaction works correctly.

Problem: Incomplete Transcription

Possible Cause: Incorrect linearized template.
Solution:  Confirm the sequence and restriction sites. Check an aliquot of purified DNA on an agarose gel.

Possible Cause: Degradation of RNA sample buffer.
Solution:  Avoid multiple freeze-thaw cycles and use newly-made buffers.

Possible Cause: Nucleotide concentration is too low.
Solution:  The concentration should always be at least 12 µM. Furthermore, adding “cold” rNTPs can increase the proportion of full-length transcripts.

Possible Cause: The reaction is terminating prematurely due to a GC-rich template.
Solution: Decrease the temperature of the transcription reaction from 37 °C to 30 °C for full-length transcripts.

Problem: Transcripts Longer than Expected

Possible Cause: Plasmid is non-linearized.
Solution: After linearization of your template, check an aliquot on an agarose gel to confirm that the digestion was complete.

Possible Cause: rUTP concentration is too high.
Solution: Decrease the concentration of the nucleotide.

Possible Cause: The template has a 3' overhang.
Solution: Use a restriction enzyme that produces 5' overhangs or blunt-ended fragments.

Planning to repeat this experiment? Consider these tips!

  • Check your template DNA is linearized via agarose gel electrophoresis
  • Use high quality DNA templates,
  • Use the recommended nucleotide concentration,
  • Always use a positive control.

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