First Strand Synthesis with Reverse Transcriptase (M0253)

New England  Biolabs

Feb 15, 2022

Version 2

First Strand Synthesis with Reverse Transcriptase (M0253) V.2

DOI

dx.doi.org/10.17504/protocols.io.bddyi27w

New England Biolabs¹

¹New England Biolabs

New England Biolabs (NEB)
Tech. support phone: +1(800)632-7799 email: info@neb.com

New England Biolabs

DOI: dx.doi.org/10.17504/protocols.io.bddyi27w

External link: https://www.neb.com/protocols/0001/01/01/first-strand-synthesis-protocol-with-reverse-transcriptase

Protocol Citation: New England Biolabs 2022. First Strand Synthesis with Reverse Transcriptase (M0253). protocols.io https://dx.doi.org/10.17504/protocols.io.bddyi27wVersion created by New England Biolabs

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: March 08, 2020

Last Modified: February 15, 2022

Protocol Integer ID: 33944

Keywords: M-MuLV reverse transcriptase, first strand reverse transcription, transcription

Abstract

This protocol is for First Strand Synthesis with M-MuLV Reverse Transcriptase (M0253).

Materials

MATERIALS
ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010 
ReagentDTT (Dithiothreitol) (> 99% pure) Protease freeGold BiotechnologyCatalog #DTT 
ReagentTris-HClLife TechnologiesCatalog #AM9855 
ReagentPotassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333  
ReagentM-MuLV Reverse TranscriptaseNew England BiolabsCatalog #M0253 

Materials needed:

10X RT buffer: 
AB
Tris-HCl (pH 8.3 @ 25°C)500 mM
KCl750 mM
MgCl230 mM
DTT100 mM
 dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)

Oligo-dT primer (40 µM)

Random nonamers (40 µM)

RNase Inhibitor (10 U/µL)

M-MuLV Reverse Transcriptase (200 units/µL)

Safety warnings

Please refer to the Safety Data Sheets (SDS) for health and environmental hazards. 

Before start

Prepare the following solutions:

10X RT buffer: 
AB
Tris-HCl (pH 8.3 @ 25°C)500 mM
KCl750 mM
MgCl230 mM
DTT100 mM
 
dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)

In a sterile microfuge tube add the following:  
ReagentVolume
RNA solution0.5-2 µg (total RNA) or 50-100 ng (PolyA-selected RNA)
Primer (Oligo-dT primer at 40 µM or Random nonamer N9 at 40 µM)2 µL
dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)4 µL
nuclease-free H2Oto final volume of 16 µL

Heat for 3-5 minutes at Temperature65 °C  -Temperature80 °C  .

Spin briefly and place promptly TemperatureOn ice  .

Add:
Amount2 µL 10X RT buffer  
Amount1 µL RNAse inhibitor  
Amount1 µL M-MuLV Reverse Transcriptase  

Final volume: Amount20 µL  

Incubate at Temperature42 °C   for Duration01:00:00  .

Inactivate enzyme at Temperature90 °C   for Duration00:10:00  .

Store products at Temperature-20 °C   or proceed to next step(s).

	A	B
	Tris-HCl (pH 8.3 @ 25°C)	500 mM
	KCl	750 mM
	MgCl2	30 mM
	DTT	100 mM

	Reagent	Volume
	RNA solution	0.5-2 µg (total RNA) or 50-100 ng (PolyA-selected RNA)
	Primer (Oligo-dT primer at 40 µM or Random nonamer N9 at 40 µM)	2 µL
	dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)	4 µL
	nuclease-free H2O	to final volume of 16 µL

Public workspaceFirst Strand Synthesis with Reverse Transcriptase (M0253) V.2

First Strand Synthesis with Reverse Transcriptase (M0253) V.2