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First Strand cDNA Synthesis Protocols (E6300) - NEB

The document provides instructions for a first strand cDNA synthesis protocol using M-MuLV reverse transcriptase. It details the components and steps to carry out the protocol, including mixing RNA and primer, denaturing RNA, adding reaction mix and enzyme, incubating, inactivating the enzyme, and storing the cDNA product.

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Aldwin Adiong
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62 views

First Strand cDNA Synthesis Protocols (E6300) - NEB

The document provides instructions for a first strand cDNA synthesis protocol using M-MuLV reverse transcriptase. It details the components and steps to carry out the protocol, including mixing RNA and primer, denaturing RNA, adding reaction mix and enzyme, incubating, inactivating the enzyme, and storing the cDNA product.

Uploaded by

Aldwin Adiong
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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First Strand cDNA Synthesis Protocols (E6300)


Introduction
Thaw system components and put on ice. A control reaction without reverse transcriptase is recommended to examine the DNA contamination in the samples.

Protocol
1. Mix RNA sample and primer d(T)23VN in two sterile RNase-free microfuge tubes.

Total RNA 1–6 μl

d(T)23VN (50 μM) 2 μl

nuclease-free H20 variable

Total Volume 8 μl

2. Denature RNA for 5 minutes at 70°C. Spin briefly and put promptly on ice. This step is optional. However, it improves the cDNA yield for long messenger RNAs and GC-rich
RNA regions.
3. Add the following components to one tube.

M-MuLV Reaction Mix 10 μl

M-MuLV Enzyme Mix 2 μl

To the negative control tube, add the following:

M-MuLV Reaction Mix 10 μl

H2O 2 μl

4. Incubate the 20 μl cDNA synthesis reaction at 42°C for one hour. If Random Primer Mix is used, an incubation step at 25°C for 5 min is recommended before the 42°C
incubation.
5. Inactivate the enzyme at 80°C for 5 minutes. Dilute reaction to 50 μl with 30 μl H2O for PCR. The cDNA product should be stored at -20°C. For downsteam PCR amplification,
the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.

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Product Categories:
RT-PCR Products, Reverse Transcriptases & RT-PCR Products, cDNA Synthesis & Reverse Transcriptases Products

Applications:
cDNA Synthesis, Reverse Transcription (cDNA Synthesis), RT-PCR & cDNA Synthesis,
| More +

Related Products:
ProtoScript® First Strand cDNA Synthesis Kit

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