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The following reagents are supplied with this product:
NEB # | Component Name | Component # | Stored at (°C) | Amount | Concentration |
E2040S | -20 |
CTP | N0454AVIAL | -20 | 1 x 0.1 ml | 100 mM | |
FLuc Control Template | N0426AVIAL | -20 | 1 x 0.01 ml | 0.5 µg/µl | |
UTP | N0453AVIAL | -20 | 1 x 0.1 ml | 100 mM | |
ATP | N0451AVIAL | -20 | 1 x 0.1 ml | 100 mM | |
T7 RNA Polymerase Mix | M0255AVIAL | -20 | 1 x 0.1 ml | ||
GTP | N0452AVIAL | -20 | 1 x 0.1 ml | 100 mM | |
10X T7 Reaction Buffer | B2041AVIAL | -20 | 1 x 0.1 ml | ||
Dithiothreitol (DTT) | B1222AVIAL | -20 | 1 x 0.5 ml | 100 mM |
E2040L | -20 |
10X T7 Reaction Buffer | B2041AAVIAL | -20 | 1 x 0.5 ml | 10 X | |
T7 RNA Polymerase Mix | M0255LVIAL | -20 | 1 x 0.5 ml | ||
FLuc Control Template | N0426AVIAL | -20 | 1 x 0.01 ml | 0.5 µg/µl | |
ATP | N0451AAVIAL | -20 | 1 x 0.5 ml | 100 mM | |
GTP | N0452AAVIAL | -20 | 1 x 0.5 ml | 100 mM | |
UTP | N0453AAVIAL | -20 | 1 x 0.5 ml | 100 mM | |
CTP | N0454AAVIAL | -20 | 1 x 0.5 ml | 100 mM | |
Dithiothreitol (DTT) | B1222AVIAL | -20 | 1 x 0.5 ml | 100 mM |
Now includes separate tube of DTT
The following reagents are supplied with this product:
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.
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).
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.
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).
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.
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.
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.
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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.
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.