Troubleshooting
Control Reaction
The CLuc control template DNA is a linearized plasmid containing the Cypridina
luciferase gene under the transcriptional control of the T7 promoter. The size of
the runoff transcript is 1.6 kb. The control reaction should yield
≥ 15 μg RNA transcript in 30 minutes.
If the control reaction is not working, there may be technical problems during reaction set up. Repeat the reaction by following the protocol carefully; take all precautions to avoid RNase contamination. Contact NEB for technical assistance.
The control plasmid sequence can be found within the DNA Sequences and Maps Tool under the name “pCMV-CLuc 2”. The CLuc control template is generated by linearizing the plasmid with restriction enzyme Xba I.
Low Yield of Full-length RNA
If the transcription reaction with your template generates full-length RNA, but
the yield is significantly lower than expected, it is possible that contaminants in
the DNA template are inhibiting the RNA polymerase, or the DNA concentration
may be incorrect. Alternatively, additional purification of DNA template may be
required. Phenol:chloroform extraction is recommended (see template DNA
preparation section).
Low Yield of Short Transcript
High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to
16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield. Alternatively, clean up the DNA template using a spin column based
method, Monarch PCR & DNA Cleanup Kit (5 μg), NEB #T1030.
RNA Transcript Smearing on Denaturing
Gel
If the RNA appears degraded (e.g., smeared) on denaturing agarose or
polyacrylamide gel, the DNA template is likely contaminated with RNase.
DNA templates contaminated with RNase can affect the length and yield of RNA
synthesized (a smear below the expected transcript length). If the plasmid DNA
template is contaminated with RNase, perform phenol:chloroform extraction,
then ethanol precipitate and dissolve the DNA in nuclease-free water (see template DNA preparation section).
RNA Transcript of Larger Size than Expected
If the RNA transcript appears larger than expected on a denaturing gel, plasmid DNA may be incompletely digested. Even small amounts of undigested
circular plasmid DNA can produce large amounts of long transcripts. Check
template for complete digestion. If undigested plasmid is confirmed, repeat
restriction enzyme digestion.
Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structure.
RNA Transcript of Smaller Size than Expected
If denaturing gel analysis shows the presence of smaller bands than the expected size, it is most likely due to premature termination by the polymerase.
Sequences with resemblance to T7 RNA Polymerase termination signals will
cause premature termination. Incubating the transcription reaction at lower
temperatures, for example at 30°C, may increase the proportion of full-length
transcript, however the yield will be decreased. For GC rich templates, or
templates with secondary structures, incubation at 42°C may improve yield of
full-length transcript.
Tailing Length Control
Tail length is defined by the poly(A) coding length on the DNA template.
Poly(A) tails longer than 125 nt have minimal effect on enhancing mRNA function.
mRNA not Functional
- Verify the mRNA is intact, capped and tailed.
- Be sure the mRNA is clean, free from any inhibitors of downstream experiments.
- Follow instructions carefully with appropriate controls.
- Verify the DNA template has the correct sequence.