Nov 02, 2022

Public workspacecDNA protocol - Thermo Scientific RevertAid RT

DOI
dx.doi.org/10.17504/protocols.io.36wgq4zwxvk5/v1
  • 1University of Oregon
Jocelynz
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DOI: dx.doi.org/10.17504/protocols.io.36wgq4zwxvk5/v1
Protocol Citation: Lauren Ponisio, Jocelyn Zorn 2022. cDNA protocol - Thermo Scientific RevertAid RT . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4zwxvk5/v1
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: September 02, 2021
Last Modified: November 02, 2022
Protocol Integer ID: 52942
Funders Acknowledgement:
Foundation of Food and Agricultural Research
Grant ID: CA18-SS-0000000009
Abstract
cDNA protocol - Thermo Scientific RevertAid RT
Protocol materials
ReagentRandom Hexamer PrimerThermo FisherCatalog #SO142
Step 5
ReagentRiboLock RNase InhibitorThermo ScientificCatalog #EO0381
Step 8
ReagentdNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
Step 9
ReagentRevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0441
Step 10
Prepare workspace
Prepare workspace
Thaw RNA and reagents on ice. Label four foil lids: one to replace RNA lid, one for reaction and two for aliquots
UV sterilize 3 RNase free PCR plates, 8 boxes 10uL filter tips, and DEPC treated H2O
Once thawed, flash spin down RNA in plate centrifuge and spin down reagents in mini centrifuge
cDNA conversion
cDNA conversion
Add Amount1 µL RNA (Amount.1 ng -Amount5 µg ) to empty PCR plate

Add Amount1 µL ReagentRandom Hexamer PrimerThermo FisherCatalog #SO142

Note
If not using ReagentRandom Hexamer PrimerThermo FisherCatalog #SO142 protocol will be different. See product handbookReagentRevertAid RT Reverse Transcription KitThermo FisherCatalog #K1691


Add Amount10 µL DEPC treated H2O
Note
If used amount other than Amount1 µL RNA (step 1), adjust H2O volume to bring total reaction volume to Amount12 µL



Add Amount4 µL 5X reaction buffer

Add Amount1 µL ReagentRiboLock RNase InhibitorThermo ScientificCatalog #EO0381 (20 U/uL)

Add Amount2 µL ReagentdNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191

Add Amount1 µL ReagentRevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0441

Note
Total reaction volume should be Amount20 µL


Mix gently with pipette and centrifuge briefly on "short"
Place in thermocycler and run program "cDNA"
Note
Thermocycler cDNA program: Lid 105°C. 25°C 5 min, 42°C 60 min, 70°C for 5 min, ∞ hold 4°C.

Aliquot product into microplates and seal with labeled foil lid
Store at Temperature-20 °C for up to 1 week or Temperature-80 °C for longer term storage