May 16, 2020

Public workspaceProtocol for Standard RNA Synthesis with Hi-T7 RNA Polymerase (NEB #M0658) V.1

DOI
https://dx.doi.org/10.17504/protocols.io.8tnhwme
Protocol for Standard RNA Synthesis with Hi-T7 RNA Polymerase (NEB #M0658)
  • New England Biolabs1
  • 1New England Biolabs
New England Biolabs
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DOI: https://dx.doi.org/10.17504/protocols.io.8tnhwme
External link: https://www.neb.com/protocols/2018/06/21/protocol-for-standard-rna-synthesis-with-hi-t7-rna-polymerase-neb-m0658
Protocol Citation: New England Biolabs 2020. Protocol for Standard RNA Synthesis with Hi-T7 RNA Polymerase (NEB #M0658). protocols.io https://dx.doi.org/10.17504/protocols.io.8tnhwme
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 28, 2019
Last Modified: May 16, 2020
Protocol Integer ID: 29262
Keywords: protocol for standard rna synthesis, t7 rna polymerase, standard rna synthesis, rna
Materials
STEP MATERIALS
ReagentNuclease-free WaterNew England BiolabsCatalog #E6317
ReagentNTPs
ReagentTemplate DNA
ReagentHi-T7 RNA Polymerase 50 U/mlNew England BiolabsCatalog #M0658S
ReagentRNase Inhibitor
Reagent10X Hi-T7 RNA Polymerase Reaction BufferNew England BiolabsCatalog #M0658S
Protocol materials
Reagent10X Hi-T7 RNA Polymerase Reaction BufferNew England BiolabsCatalog #M0658S
ReagentNuclease-free WaterNew England BiolabsCatalog #E6317
ReagentNTPs
ReagentTemplate DNA
ReagentHi-T7 RNA Polymerase 50 U/mlNew England BiolabsCatalog #M0658S
ReagentRNase Inhibitor
Troubleshooting
Safety warnings
For safety information and warnings, please refer to the SDS (Safety Data Sheet).
Assemble the reaction
Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottom of the tubes.
Assemble the reaction at TemperatureRoom temperature in the following order.
Note
The total reaction volume is Amount20 µL .

Add Nuclease-free H2O: X µl
ReagentNuclease-free WaterNew England BiolabsCatalog #E6317

Add 10X Hi-T7 RNA Polymerase Reaction Buffer: Amount2 µL Concentration1 X
Reagent10X Hi-T7 RNA Polymerase Reaction BufferNew England BiolabsCatalog #M0658S

Note
Hi-T7 RNA Polymerase Supplied with 10X Hi-T7 RNA Polymerase Reaction Buffer formulated for optimal performance.

Add NTPs: X µl Concentration0.5 millimolar (mM) each
ReagentNTPs

Add Template DNA: X µl from Concentration0.2 µg to Concentration1 µg
ReagentTemplate DNA

Add RNase Inhibitor: Amount0.5 µL Concentration1 U/µl final
ReagentRNase Inhibitor

Optional
Add Hi-T7 RNA Polymerase (50 U/µl): Amount2 µL
ReagentHi-T7 RNA Polymerase 50 U/mlNew England BiolabsCatalog #M0658S

Incubation
Incubate at Temperature50 °C for Duration01:00:00 .

Incubation