Nov 02, 2022
cDNA protocol - Thermo Scientific RevertAid RT
- 1University of Oregon
- FFAR
- Ponisiolab
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
Keywords: cdna protocol, thermo scientific revertaid rt, protocol
Funders Acknowledgements:
Foundation of Food and Agricultural Research
Grant ID: CA18-SS-0000000009
Abstract
cDNA protocol - Thermo Scientific RevertAid RT
Protocol materials
Random Hexamer PrimerThermo FisherCatalog #SO142
RiboLock RNase InhibitorThermo ScientificCatalog #EO0381
dNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
RevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0441
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
Add 1 µL RNA (.1 ng -5 µg ) to empty PCR plate
Add 1 µL Random Hexamer PrimerThermo FisherCatalog #SO142
Note
If not using Random Hexamer PrimerThermo FisherCatalog #SO142 protocol will be different. See product handbookRevertAid RT Reverse Transcription KitThermo FisherCatalog #K1691
Add 10 µL DEPC treated H2O
Note
If used amount other than 1 µL RNA (step 1), adjust H2O volume to bring total reaction volume to 12 µL
Add 4 µL 5X reaction buffer
Add 1 µL RiboLock RNase InhibitorThermo ScientificCatalog #EO0381 (20 U/uL)
Add 2 µL dNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
Add 1 µL RevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0441
Note
Total reaction volume should be 20 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 -20 °C for up to 1 week or -80 °C for longer term storage