The HiScribe T7 High Yield RNA Synthesis Kit is an extremely flexible system for in vitro transcription of RNA using T7 RNA Polymerase. The kit allows for synthesis many kinds of RNA including internally labeled and co-transcriptionally capped transcripts.
RNA synthesized from the kit is suitable for many applications including RNA structure and function studies, ribozyme biochemistry, probes for RNase protection assays and hybridization based blots, anti-sense RNA and RNAi experiments, microarray analysis, microinjection, and in vitro translation and RNA vaccines.
The kit contains sufficient reagents for 50 reactions of 20 μl each. Each standard reaction yields up to 180 μg of RNA from 1 μg control template. Each kit can yield up to 9 mg RNA. For 32P labeling, the kit contains enough reagents for 100 reactions of 20 μl each.
Materials Not Included:
DNA Template: The DNA template must be linear and contain the T7 RNA Polymerase promoter with correct orientation in relation to target sequence to be transcribed.
3′-O-Me-m7G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1411)
m7G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1404)
m7G(5′)ppp(5′)A RNA Cap Structure Analog (NEB #S1405)
G(5′)ppp(5′)A RNA Cap Structure Analog (NEB #S1406)
G(5′)ppp(5′)G RNA Cap Structure Analog (NEB #S1407)
Modified-NTP: Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP
Purification: Buffer- or water-saturated phenol/chloroform, ethanol and 3 M sodium acetate, pH 5.2, spin columns
Gel Analysis: Gels and running buffers, gel apparatus, power supply
Figure 1. Transcription by T7 RNA Polymerase
Figure 2. Time course of standard RNA synthesis from three DNA templates
Reactions were incubated at 37°C in a PCR machine. Transcripts were purified by spin columns and quantified on NanoDrop™ Spectrophotometer.
Figure 3. Effect of template amount on RNA yield
Standard reactions were incubated at 37°Cin a PCR machine for 2 hours. Transcripts were purified by spin columns and quantified on NanoDrop™ Spectrophotometer.
Figure 4: Improved RNA yield and integrity from extended duration transcription reactions
reactions were assembled, in duplicate, according to the manufacturers’ suggested protocols using 3 ng of dsDNA template encoding a 1.8 kb RNA, and incubated at 37°C for 16, 24 and 40 hours. At each time point, the corresponding tubes were transferred to -20°C to stop the reaction. Transcription reactions were column purified after the last time point.
(A) Transcript yield – After column purification, RNA concentration was measured using a NanoDrop spectrophotometer and total RNA yield was calculated. These data demonstrate that a substantially higher yield of RNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.
(B) Transcript integrity – 150 ng of column purified RNA was run a 1.2% denaturing agarose gel, stained with ethidium bromide and visualized by UV fluorescence. The data demonstrate greatly improved transcript integrity after extended duration RNA synthesis reactions using the HiScribe T7 High Yield RNA Synthesis Kit as compared to the competitor’s kit.
This product is related to the following categories:
The FLuc control template DNA is a linearized plasmid containing the firefly luciferase gene under the transcriptional control of T7 promoter. The size of the runoff transcript is 1.8 kb. The control reaction should yield ≥ 150 μg RNA transcript in 2 hours.
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 any precaution 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 "FLuc Control Plasmid". The FLuc control template is generated by linearizing the plasmid with StuI.
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.
RNA Transcript Smearing on Denaturing Gel
If the RNA appears degraded (e.g. smeared) on denaturing agarose or polyacrylamide gel, DNA template is 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, template plasmid DNA may be incompletely digested. Even small amounts of undigested circular 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 structures.
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. Some sequences which resemble 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.
If premature termination of transcription is found in high specific activity radiolabeled RNA probe synthesis, increase the concentration of “limiting NTP”. Additional "cold" NTP can be added to the reaction to increase the proportion of full-length transcript, however the improvement in yield of full-length product will compromise the specific activity of the probe.
Tech Tips
It is important to mix each component well before setting up reactions.
Make sure reactions are thoroughly mixed.
We recommend incubating the reactions in a dry air incubator or in a PCR machine.
Lee, NC., Larionov, V., Kouprina, N. (2015) Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast Nucleic Acids Res; 43(8), e55. PubMedID: 25690893
Jaitin, DA., Kenigsberg, E., Keren-Shaul, H., Elefant, N., Paul, F., Zaretsky, I., Mildner, A., Cohen, N., Jung, S., Tanay, A. and Amit, I. (2014) Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types. Science; 343, 776-779. PubMedID: 24531970
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