cDNA synthesis
Miquel Vila
Published: 2022-09-13 DOI: 10.17504/protocols.io.kqdg3948qg25/v1
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Abstract
High-Capacity cDNA Reverse Transcription Kits(#4368814, Applied Biosystems - ThermoFisher)
Steps
cDNA synthesis
1.
Prepare the 2X master mix (ul per sample):
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2ul RT Buffer
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0.8ul 25X dNTP Mix(100mM)
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2ul Random primers
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1ul Multiscribe Reverse Transcriptase
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4.2ul Nuclease free water
2.
Take 0.5ug of RNA in a maximum of 10ul of sample volume
3.
Mix the 2X master mix (10ul) with the RNA samples (10ul) and mix by pippeting
4.
Centrifuge the plates to spin down all the reagents.
5.
Keep the samples on ice
6.
Program the thermal cycler to 25C for 10min 37C for 120 mintues 85C for 5 minutes and 4C until finished.
7.
Store the cDNA samples at -20C for short-term storage or -80C for long-term storage (years).
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