First strand cDNA synthesis (ThermoScientific RevertAid)

Ben Kuipers

Oct 16, 2019

First strand cDNA synthesis (ThermoScientific RevertAid)

Forked from First strand cDNA synthesis (ThermoScientific RevertAid)

DOI

https://dx.doi.org/10.17504/protocols.io.8bmhsk6

Ben Kuipers¹

¹Wageningen University

iGEM Wageningen 2019

Ben Kuipers

DOI: https://dx.doi.org/10.17504/protocols.io.8bmhsk6

Protocol Citation: Ben Kuipers 2019. First strand cDNA synthesis (ThermoScientific RevertAid). protocols.io https://dx.doi.org/10.17504/protocols.io.8bmhsk6

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 16, 2019

Last Modified: October 16, 2019

Protocol Integer ID: 28749

Keywords: first strand cdna synthesis, strand cdna for use, strand cdna, pcr, thermoscientific revertaid

Abstract

The following protocol is optimized to generate first-strand cDNA for use in (q)PCR

Materials

MATERIALS
Reagent5X RT BufferThermo Fisher ScientificCatalog ##B91 
ReagentdNTP Mix 10 mM eachThermo Fisher ScientificCatalog ##R0191 
ReagentWater, nuclease free 
ReagentRiboLock RNase Inhibitor Thermo Fisher ScientificCatalog ##EO0381 
ReagentRevertAid Reverse Transcriptase (200 U/&micro;L)Thermo FisherCatalog #EP0442 
ReagentOligo(dT)18 PrimerThermo FisherCatalog #SO131 

Troubleshooting

Before start

Mix and briefly centrifuge all reagents after thawing, keep on ice.

Add reaction components into sterile, nuclease-free tube on ice in the indicated order:
 
Template RNA100 ng  ( 1pg - 5 µg)
Oligo(dT)181 µl (100 pmol)
Water, nuclease-freeto 12 µl
 

Optional: If the RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65 °C for 5min. Chill on ice, briefly centrifuge again and place on ice
ice.

 
 
5X RT Buffer4 µl
RiboLock RNase Inhibitor1 µl (20 U)
RevertAid RT (200 U/µL)2 µl (400 U)
10 mM dNTP Mix1 µl
Total volume20 µl
 
Mix gently and centrifuge briefly.

 
min25 °C
min42 °C (For GC-rich RNA, the reaction temperature can be increased to 45 °C)
min70 °C

The cDNA product is now ready for downstream applications

	Template RNA	100 ng ( 1pg - 5 µg)
	Oligo(dT)18	1 µl (100 pmol)
	Water, nuclease-free	to 12 µl

	5X RT Buffer	4 µl
	RiboLock RNase Inhibitor	1 µl (20 U)
	RevertAid RT (200 U/µL)	2 µl (400 U)
	10 mM dNTP Mix	1 µl
	Total volume	20 µl

	5 min	25 °C
	60 min	42 °C (For GC-rich RNA, the reaction temperature can be increased to 45 °C)
	5 min	70 °C

Public workspaceFirst strand cDNA synthesis (ThermoScientific RevertAid)

First strand cDNA synthesis (ThermoScientific RevertAid)