First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)

New England  Biolabs

Apr 11, 2022

Version 2

First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368) V.2

DOI

dx.doi.org/10.17504/protocols.io.x14egnmmv5dy/v2

New England Biolabs¹

¹New England Biolabs

New England Biolabs

DOI: dx.doi.org/10.17504/protocols.io.x14egnmmv5dy/v2

External link: https://www.neb.com/protocols/2012/10/03/first-strand-cdna-synthesis-kit-using-protoscript-ii-reverse-transcriptase-m0368

Protocol Citation: New England Biolabs 2022. First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368). protocols.io https://dx.doi.org/10.17504/protocols.io.x14egnmmv5dy/v2Version 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 07, 2020

Last Modified: April 11, 2022

Protocol Integer ID: 33924

Keywords: murine RNAse inhibitor, transcription, Reverse transcription, RT, cDNA

Abstract

This protocol explains methods for the "First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)".

Materials

MATERIALS
ReagentRNase Inhibitor, Murine - 3,000 unitsNew England BiolabsCatalog #M0314S 
ReagentProtoScript II Reverse Transcriptase - 4,000 unitsNew England BiolabsCatalog #M0368S 
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S 
ReagentDeoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S 
0.1 M DTT

Safety warnings

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

In a sterile microfuge tube add:
AB
RNA solution 1ng-1 µg total RNA or 1-100 ng polyA-selected RNA Primer  (50 µM dT or 60 µM Random Primer Mix)2 µl
10 mM dNTP mix1 µl
Nuclease-free H2Oto final volume of 12 µl

Optional: Heat for 3-5 minutes at Temperature65 °C  -Temperature70 °C  . Spin briefly and place promptly TemperatureOn ice  .

Add the following (bringing to 20 µl final volume):
AB
5X ProtoScript II RT Reaction buffer4 µl
0.1 M DTT2 µl
Murine RNase inhibitor (40 U/µl)1 µl
ProtoScript II Reverse Transcriptase (200 U/µl)1 µl
Final volume20 µl

If Random Primer Mix is used, an incubation step at Temperature25 °C   for Duration00:05:00   is recommended before the Temperature42 °C   incubation in the next step.

Incubate at Temperature42 °C   for 30 minutes-1 hour.

Inactivate enzyme at Temperature80 °C   for Duration00:05:00  .

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

	A	B
	RNA solution 1ng-1 µg total RNA or 1-100 ng polyA-selected RNA Primer (50 µM dT or 60 µM Random Primer Mix)	2 µl
	10 mM dNTP mix	1 µl
	Nuclease-free H2O	to final volume of 12 µl

	A	B
	5X ProtoScript II RT Reaction buffer	4 µl
	0.1 M DTT	2 µl
	Murine RNase inhibitor (40 U/µl)	1 µl
	ProtoScript II Reverse Transcriptase (200 U/µl)	1 µl
	Final volume	20 µl

Public workspaceFirst Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368) V.2

First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368) V.2