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library PREPARATION

NEBNext Poly(A) mRNA Magnetic Isolation Module


Instruction Manual

NEB #E7490S/L 24/96 reactions

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NEBNext Poly(A) mRNA Magnetic Isolation Module

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

The NEBNext Poly(A) mRNA Magnetic Isolation Module Includes:


The volumes provided are sufficient for preparation of up to 24 reactions (NEB#E7490S) and 96reactions (NEB #E7490L). All reagents should be stored at 4C.

NEBNext Oligo d(T)25 Beads NEBNext RNA Binding Buffer (2X) NEBNext Wash Buffer NEBNext Elution Buffer Nuclease-free Water

Required Materials Not Included:


96-well 0.2 ml PCR Plates and Microseal 'B' Adhesive Sealer (BioRad MSB-1001) or 0.2 ml RNase-free tube.
ISO 9001
Registered

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.

Isolate mRNA using the NEBNext Oligo d(T)25 Magnetic Beads:


Starting Material: 15 g of total RNA. 1. Dilute the total RNA with nuclease-free water to a final volume of 50 l in a nuclease-free 0.2 ml PCR tube. 2. Aliquot 15 l of NEBNext Magnetic Oligo d(T)25 Beads into nuclease-free 0.2 ml PCR tube. 3. Wash the beads two times with 100 l of 2X RNA Binding Buffer and remove the supernatant. 4. Resuspend the beads in 50 l of 2X RNA Binding Buffer and add the 50 l of total RNA sample from step 1. 5. Place the tubes on the thermal cycler and heat the sample at 65C for 5 minutes and hold at 4C to denature the RNA and facilitate binding of the poly-A-RNA to the beads. 6. Remove tubes from the thermal cycler when the temperature reaches 4C. 7. Place the tubes on the bench and incubate at room temperature for 5 minutes to allow the RNA to bind to the beads. 8. Place the tubes on the magnetic rack at room temperature for 2 minutes to separate the poly-A RNA bound to the beads from the solution. 9. Remove and discard all of the supernatant. Take care not to disturb the beads. 10. Remove the plate from the magnetic rack. 11. Wash the beads twice with 200 l of Wash Buffer to remove unbound RNA. Pipette the entire volume up and down 6 times to mix thoroughly. 12. Place the tubes on the magnetic rack at room temperature for 2 minutes. 13. Remove and discard all the supernatant from each well of the plate using a multichannel pipette. Take care not to disturb the beads. 14. Remove the tubes from the magnetic rack. 15. Add 50 l of elution buffer to each well of the plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. 16. Place the tubes on the thermal cycler. Close the lid and heat the sample at 80C for 2 minutes, then hold at 25C to elute the poly-A RNA from the beads.

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.

Figure 2: Example of mRNA distribution on a bioanalyzer.


[FU] 8 7 6 5 4 3 2 1 0 20 30 40 50 60 70 80 [s]

NEBNext Oligo d(T)25 Beads


#E7491A: 0.360 ml #E7491AA: 1.44 ml Store at 4C Description: Oligo d(T)25 Magnetic Beads are a 1 m polymer based affinity matrix for the small-scale isolation of mRNA from previously isolated total RNA. The isolation occurs through the hybridization of covalently coupled oligo d(T)25 to the poly(A) region present in most eukaryotic mRNA. The magnetic separation technology is scalable and permits elution of intact mRNA in small volumes eliminating the need for precipitation of the isolated mRNA. Support Matrix: 1 m nonporous superparamagnetic microparticles Diameter: 1 M Surface area: 3038 m2/g Magnetic mass susceptibility: 44 emu/g Lot Controlled

NEBNext RNA Binding Buffer (2X)


#E7492A: #E7492AA: Store at 4C 1X NEBNext RNA Binding Buffer: 1 M LiCl 40 mM Tris HCl (pH 7.5) 2 mM EDTA 0.1% Triton X-100 7.2 ml 28.8 ml

Quality Control Assays


Endonuclease Activity: Incubation of a 50 l reaction with the 2X Binding buffer and 1 g of X174 RF 1 DNA for 4 hours at 37C results in less than 10% conversion to RF II as determined by agarose gel electrophoresis. Phosphatase Activity: Incubation of a minimum of 10 l of the 2X Binding Buffer at a 1X concentration in protein phosphatase assay buffer (1M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) containing 2.5 mM p-nitrophenyl phosphate at 37C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm. 16-Hour Incubation: 50 l reactions containing the 2X Binding Buffer at 1X concentration and 1g of HindIII digested Lambda DNA incubated for 16 hours at 37C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 l reactions containing this reaction buffer at 1X concentration and 1 g T3 DNA incubated for 16 hours at 37C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. RNase Activity: Incubation of the 2X Binding Buffer at a 1X concentration with 40 ng of a FAM-labeled RNA transcript for 16 hours at 37C results in no detectable RNase Activity as determined by polyacrylamide gel electrophoresis. Lot Controlled

NEBNext Wash Buffer


#E7493A: 28.8 ml #E7493AA: 115.2 ml Store at 4C 1X NEBNext Wash Buffer: 150 mM LiCl 20 mM Tris HCl (pH 7.5) 1.0 mM EDTA 0.01% Triton X-100

NEBNext Elution Buffer


#E7494A: 6.0 ml #E7494AA: 24.0 ml Store at 4C 1X NEBNext Elution Buffer: 10 mM Tris HCl (pH 7.5)

Quality Control Assays


Endonuclease Activity: Incubation of a 50 l reaction with Elution buffer and 1g of X174 RF I DNA for 4 hours at 37C results in less than 10% conversion to RF II as determined by agarose gel electrophoresis. Phosphatase Activity: Incubation of a minimum of 10 l of the Elution Buffer at a 1X concentration in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) containing 2.5 mM p-nitrophenyl phosphate at 37C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm. 16-Hour Incubation: 50 l reactions containing the Elution Buffer at 1X concentration and 1 g of HindIII digested Lambda DNA incubated for 16 hours at 37C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 l reactions containing the Elution buffer at 1X concentration and 1 g T3 DNA incubated for 16 hours at 37C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. RNase Activity: Incubation of the Elution Buffer at a 1X concentration with 40ng of a FAM-labeled RNA transcript for 16 hours at 37C results in no detectable RNase Activity as determined by polyacrylamide gel electrophoresis. Lot Controlled

Quality Control Assays


Endonuclease Activity: Incubation of a 50 l reaction with Wash buffer and 1 g of X174 RF I DNA for 4 hours at 37C results in < 10% conversion to RF II as determined by agarose gel electrophoresis. Phosphatase Activity: Incubation of a minimum of 10 l of the Wash Buffer at a 1X concentration in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) containing 2.5 mM p-nitrophenyl phosphate at 37C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm. 16-Hour Incubation: 50 l reactions containing the Wash Buffer at 1X concentration and 1 g of HindIII digested Lambda DNA incubated for 16 hours at 37C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 l reactions containing this reaction buffer at 1X concentration and 1 g T3 DNA incubated for 16 hours at 37C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. RNase Activity: Incubation of the Wash Buffer at a 1X concentration with 40 ng of a FAM-labeled RNA transcript for 16 hours at 37C results in no detectable RNase Activity as determined by polyacrylamide gel electrophoresis. Lot Controlled

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.

Quality Control Assays


16-Hour Incubation: 50 l reactions containing Nuclease-free Water and 1 g of HindIII digested Lambda DNA incubated for 16 hours at 37C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 l reactions containing Nuclease-free Water and 1 g of T3 DNA incubated for 16 hours at 37C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. Endonuclease Activity: Incubation of a 50 l reaction containing Nucleasefree Water with 1 g of X174 RF I DNA for 4 hours at 37C results in < 10% conversion to RF II as determined by gel electrophoresis. RNase Activity: Incubation of a 10 l reaction containing Nuclease-free Water with 40 ng of a FAM-labeled RNA transcript for 16 hours at 37C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis. Phosphatase Activity: Incubation of a minimum of 10 l of nuclease free water at a 1X concentration in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) containing 2.5 mM p-nitrophenyl phosphate at 37C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm. Lot Controlled

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DNA Cloning DNA Amplification & PCR Epigenetics RNA Analysis Library Prep for Next Gen Sequencing Protein Expression & Analysis Cellular Analysis

USA New England Biolabs, Inc. 240 County Road Ipswich, MA 01938-2723 Telephone: (978) 927-5054 Toll Free: (USA Orders) 1-800-632-5227 Toll Free: (USA Tech) 1-800-632-7799 Fax: (978) 921-1350 e-mail: info@neb.com www.neb.com Canada New England Biolabs, Ltd. Telephone: (905) 837-2234 Toll Free: 1-800-387-1095 Fax: (905) 837-2994 Fax Toll Free: 1-800-563-3789 e-mail: info@ca.neb.com China, Peoples Republic New England Biolabs (Beijing), Ltd. Telephone: 010-82378265/82378266 Fax: 010-82378262 e-mail: info@neb-china.com

France New England Biolabs France Free Call: 0800/100 632 Free Fax: 0800/100 610 e-mail: info@fr.neb.com Germany New England Biolabs GmbH Telephone: +49/(0)69/305 23140 Free Call: 0800/246 5227 (Germany) Fax +49/(0)69/305 23149 Free Fax: 0800/246 5229 (Germany) e-mail: info@de.neb.com Japan New England Biolabs Japan, Inc. Telephone: +81 (0)3 5669 6191 Fax +81 (0)3 5669 6192 e-mail: info@neb-japan.com United Kingdom New England Biolabs (UK), Ltd. Telephone: (01462) 420616 Call Free: 0800 318486 Fax: (01462) 421057 Fax Free: 0800 435682 e-mail: info@uk.neb.com

Version 1.1 8/12

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