cDNA protocol - Thermo Scientific RevertAid RT
Lauren Ponisio, Jocelyn Zorn
Published: 2022-11-03 DOI: 10.17504/protocols.io.36wgq4zwxvk5/v1
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Abstract
cDNA protocol - Thermo Scientific RevertAid RT
Steps
Prepare workspace
1.
Thaw RNA and reagents on ice. Label four foil lids: one to replace RNA lid, one for reaction and two for aliquots
2.
UV sterilize 3 RNase free PCR plates, 8 boxes 10uL filter tips, and DEPC treated H2O
3.
Once thawed, flash spin down RNA in plate centrifuge and spin down reagents in mini centrifuge
cDNA conversion
4.
Add 1µL RNA (.1ng -5µg) to empty PCR plate
5. If not using Random Hexamer Primer protocol will be different. See product handbookRevertAid RT Reverse Transcription Kit
Add 1µL
Note
6. If used amount other than 1µL RNA (step 1), adjust H2O volume to bring total reaction volume to 12µL
Add 10µL DEPC treated H2O
Note
7.
Add 4µL 5X reaction buffer
8.
Add 1µL
9.
Add 2µL
10. Total reaction volume should be 20µL
Add 1µL
Note
11.
Mix gently with pipette and centrifuge briefly on "short"
12. Thermocycler cDNA program: Lid 105°C. 25°C 5 min, 42°C 60 min, 70°C for 5 min, ∞ hold 4°C.
Place in thermocycler and run program "cDNA"
Note
13.
Aliquot product into microplates and seal with labeled foil lid
14.
Store at -20°C for up to 1 week or -80°C for longer term storage
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