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Table of Contents:
Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 NEBNext Oligo d(T)25 Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 NEBNext RNA Binding Buffer (2X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 NEBNext Wash Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 NEBNext Elution Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Nuclease-free Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
NEBNext Oligo d(T)25 Beads NEBNext RNA Binding Buffer (2X) NEBNext Wash Buffer NEBNext Elution Buffer Nuclease-free Water
ISO 14001
Registered
ISO 13485
Registered
Magnetic Rack (Alpaqua, cat #A001322 or equivalent) Thermal cycler or heat block
Quality Management
Environmental Management
Medical Devices
NEW ENGLAND BIOLABS is a registered trademark of New England Biolabs, Inc. NEBNext is a registered trademark of New England Biolabs, Inc. BIOANALYZER is a registered trademark of Agilent Technologies, Inc. microseal is a registered trademark of BioRad.
Description:
The NEBNext Poly(A) mRNA Magnetic Isolation Module is designed to isolate intact poly(A)+ RNA from previously isolated total RNA. The technology is based on the coupling of Oligo d(T)25 to 1 m paramagnetic beads which is then used as the solid support for the direct binding of poly(A)+ RNA. Thus, the procedure permits the manual processing of multiple samples and can be adapted for automated high-throughput applications. Additionally, magnetic separation technology permits elution of intact mRNA in small volumes eliminating the need for precipitating the poly(A)+ transcripts in the eluent. Intact poly(A)+ RNA which is fully representative of the mRNA population of the original sample can be obtained in less than one hour.
Application:
Isolation of poly(A)+ RNA transcript from Total RNA for RNA library preparation and sequencing.
17. Remove the tubes from the thermal cycler when the temperature reaches 25C. 18. Add 50 l of 2X RNA binding buffer to each sample to allow the RNA to bind to the beads. Gently pipette the entire volume up and down 6 times to mix thoroughly. 19. Incubate the tubes at room temperature for 5 minutes. 20. Place the tubes on the magnetic stand at room temperature for 2 minutes. 21. Remove and discard all of the supernatant from each tube. Take care not to disturb the beads. 22. Remove the tubes from the magnetic stand. 23. Wash the beads twice with 200 l of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly. 24. Place the tubes on the magnetic stand at room temperature for 2 minutes. 25. Remove and discard all of the supernatant from each tube. Take care not to disturb the beads. 26. Remove the tubes from the magnetic stand. 27. Elute the mRNA from the beads by adding 17 l of the Elution Buffer and incubating the sample at 80C for 2 minutes. Immediately, place the tubes on the magnetic rack. 28. Collect the purified mRNA by transferring the supernatant to a clean nuclease-free PCR Tube. 29. Place tube on ice. 30. Assess the Yield and the Size Distribution of the purified mRNA. Run 1 l on the Bioanalyzer (Agilent Technologies, Inc.) using a RNA Pico Chip.
Nuclease-free Water
#E7495A: 1.2 ml #E7495AA: 4.8 ml Store at 20C or 4C Description: Nuclease-free Water is free of detectable DNA and RNA nucleases and phosphatases and suitable for use in DNA and RNA applications.
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DNA Cloning DNA Amplification & PCR Epigenetics RNA Analysis Library Prep for Next Gen Sequencing Protein Expression & Analysis Cellular Analysis
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