Protocol for Use with Previously Fragmented RNA - NEBNext mRNA First Strand Synthesis Module (E7525)
Protocol B for Use with Previously Fragmented RNA
Starting Material: 100–250 ng of purified mRNA fragmented to 200 nt and cleaned up in 13.5 µl of nuclease free water
- First Strand cDNA Synthesis (Non-Directional Reaction Setup)
Mix the following components in a sterile PCR tube
Component Volume Fragmented mRNA 13.5 µl (pink) Random Primers 1 µl Total Volume 14.5 µl
- Incubate in a preheated thermocycler for 5 minutes at 65°C with heated lid set to 105°C. Hold at 4°C.
- Spin tube briefly and place on ice 8
- To the fragmented mRNA and Random Primers add:
Component Volume (pink) First Strand Synthesis Reaction Buffer 4 µl (pink) Murine RNase Inhibitor 0.5 µl Total Volume 19 µl
- Incubate in a preheated thermocycler for 2 minutes at 25°C.
- Add 1 µl ProtoScript II Reverse Transcriptase to the reaction.
- Incubate the samples in a preheated thermal cycler (with the heated lid set to ≥ 80°C)
10 minutes at 25°C
50 minutes at 42°C
15 minutes at 70°C
Hold at 4°C
- Place the tube on ice.
- Proceed directly to second strand synthesis using NEBNext mRNA Second Strand Synthesis Module (NEB #E6111).