neb mrna lisolation manual

7/28/2019 NEB mRNA Lisolation Manual

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Instuction Mnu

lIbrary PrEParaTION

NEbNext

Po(a) mrNa

Mgnetic Isotion Modue

NEb #E7490S/l

24/96 ections


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1

Te of Contents:

Desription 2

Appliation 2

Protool 3

NEBNext Oligo d(T)25

Beads 6

NEBNext RNA Binding Buer (2X) 7

NEBNext Wash Buer 8

NEBNext Elution Buer 9

Nulease-ree Water 10

The NEbNext Po(a) mrNa

Mgnetic Isotion Modue Incudes:The volumes provided are sufcient or preparation o up to 24 reactions (NEB #E7490S)

and 96reactions (NEB #E7490L). All reagents should be stored at 4C.

NEbNext Oigo d(T)25

beds

NEbNext rNa binding buffe (2X)NEbNext Wsh buffe

NEbNext Eution buffe

Nucese-fee Wte

requied Mteis Not Incuded:96-we 0.2 m PCr Ptes nd Micose 'b' adhesive See (biord MSb-1001)

o 0.2 m rNse-fee tue.

Mgnetic rck (apqu, ct #a001322 o equivent)

Them cce o het ock

NEbNext Po(a) mrNaMgnetic Isotion Modue

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BIOANALYZER is a registered tradeark o Agilent Tehnologies, In

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Desciption:The NEBNext Poly(A) RNA magneti Isolation module is designed to isolateintat poly(A)+ RNA ro previously isolated total RNA The tehnology is basedon the oupling o Oligo d(T)

25to 1 paraagneti beads whih is then used

as the solid support or the diret binding o poly(A)+ RNA Thus, the proedureperits the anual proessing o ultiple saples and an be adapted orautoated high-throughput appliations Additionally, agneti separationtehnology perits elution o intat RNA in sall volues eliinating the needor preipitating the poly(A)+ transripts in the eluent Intat poly(A)+ RNA whihis ully representative o the RNA population o the original saple an beobtained in less than one hour

appiction:Isolation o poly(A)+ RNA transript ro Total RNA or RNA library preparationand sequening

Isote mrNa using the NEbNext Oigo d(T)25

Mgnetic beds:

Starting Material: 15 g o total RNA

1 Dilute the total RNA with nulease-ree water to a fnal volue o 50 l ina nulease-ree 02 l PcR tube

2 Aliquot 15 l o NEBNext magneti Oligo d(T)25

Beads into nulease-ree02 l PcR tube

3 Wash the beads two ties with 100 l o 2X RNA Binding Buer andreove the supernatant

4 Resuspend the beads in 50 l o 2X RNA Binding Buer and add the 50 lo total RNA saple ro step 1

5 Plae the tubes on the theral yler and heat the saple at 65c or 5inutes and hold at 4c to denature the RNA and ailitate binding o thepoly-A-RNA to the beads

6 Reove tubes ro the theral yler when the teperature reahes 4c

7 Plae the tubes on the benh and inubate at roo teperature or 5inutes to allow the RNA to bind to the beads

8 Plae the tubes on the agneti rak at roo teperature or 2 inutesto separate the poly-A RNA bound to the beads ro the solution

9 Reove and disard all o the supernatant Take are not to disturb thebeads

10 Reove the plate ro the agneti rak11 Wash the beads twie with 200 l o Wash Buer to reove unbound

RNA Pipette the entire volue up and down 6 ties to ix thoroughly

12 Plae the tubes on the agneti rak at roo teperature or 2 inutes

13 Reove and disard all the supernatant ro eah well o the plate usinga ultihannel pipette Take are not to disturb the beads

14 Reove the tubes ro the agneti rak

15 Add 50 l o elution buer to eah well o the plate Gently pipette theentire volue up and down 6 ties to ix thoroughly

16 Plae the tubes on the theral yler close the lid and heat the sapleat 80c or 2 inutes, then hold at 25c to elute the poly-A RNA ro thebeads


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17 Reove the tubes ro the theral yler when the teperature reahes25c

18 Add 50 l o 2X RNA binding buer to eah saple to allow the RNA tobind to the beads Gently pipette the entire volue up and down 6 tiesto ix thoroughly

19 Inubate the tubes at roo teperature or 5 inutes

20 Plae the tubes on the agneti stand at roo teperature or 2 inutes

21 Reove and disard all o the supernatant ro eah tube Take are notto disturb the beads

22 Reove the tubes ro the agneti stand

23 Wash the beads twie with 200 l o Wash Buer Gently pipette the entirevolue up and down 6 ties to ix thoroughly

24 Plae the tubes on the agneti stand at roo teperature or 2 inutes

25 Reove and disard all o the supernatant ro eah tube Take are notto disturb the beads

26 Reove the tubes ro the agneti stand

27 Elute the RNA ro the beads by adding 17 l o the Elution Buer andinubating the saple at 80c or 2 inutes Iediately, plae the tubeson the agneti rak

28 collet the purifed RNA by transerring the supernatant to a leannulease-ree PcR Tube

29 Plae tube on ie

30 Assess the Yield and the Size Distribution o the purifed RNA Run 1 lon the Bioanalyzer (Agilent Tehnologies, In) using a RNA Pio chip

Figue 2: Exmpe of mrNa distiution on ionze.

[FU]

5

4

3

2

1

20 40 50 60 [s]

6

0

30

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70 80


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NEbNext Oigo d(T)25

beds#E7491A: 0.360 ml#E7491AA: 1.44 ml

Store at 4C

Description:

Oligo d(T)25

magneti Beads are a 1 polyer based afnity atrix or thesall-sale isolation o RNA ro previously isolated total RNA The isola-tion ours through the hybridization o ovalently oupled oligo d(T)

25to the

poly(A) region present in ost eukaryoti RNA The agneti separationtehnology is salable and perits elution o intat RNA in sall volueseliinating the need or preipitation o the isolated RNA

Support Matrix: 1 nonporous superparaagneti iropartiles

Diameter: 1 m

Surface area: 3038 2/g

Magnetic mass susceptibility: 44 eu/g

Lot Controlled

NEbNext rNa binding buffe (2X)#E7492A: 7.2 ml#E7492AA: 28.8 ml

Store at 4C

1X NEBNext RNA Binding Buffer:

1 m Licl

40 m Tris Hcl (pH 75)

2 m EDTA

01% Triton X-100

Quit Conto asss

Endonuclease Activity: Inubation o a 50 l reation with the 2X Binding buerand 1 g o X174 RF 1 DNA or 4 hours at 37c results in less than 10%onversion to RF II as deterined by agarose gel eletrophoresis

Phosphatase Activity: Inubation o a iniu o 10 l o the 2X BindingBuer at a 1X onentration in protein phosphatase assay buer (1mdiethanolaine @ pH 98 and 05 m mgcl

2) ontaining 25 m p-nitrophenyl

phosphate at 37c or 4 hours yields no detetable p-nitrophenylene anion asdeterined by spetrophotoetri analysis at 405 n

16-Hour Incubation: 50 l reations ontaining the 2X Binding Buer at 1Xonentration and 1g o HindIII digested Labda DNA inubated or 16hours at 37c results in no detetable non-speif nulease degradation asdeterined by agarose gel eletrophoresis 50 l reations ontaining thisreation buer at 1X onentration and 1 g T3 DNA inubated or 16 hoursat 37c also results in no detetable non-speif nulease degradation asdeterined by agarose gel eletrophoresis

RNase Activity: Inubation o the 2X Binding Buer at a 1X onentrationwith 40 ng o a FAm-labeled RNA transript or 16 hours at 37c results in nodetetable RNase Ativity as deterined by polyarylaide gel eletrophoresis

Lot Controlled


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NEbNext Wsh buffe#E7493A: 28.8 ml#E7493AA: 115.2 ml

Store at 4C

1X NEBNext Wash Buffer:

150 m Licl


10 m EDTA

001% Triton X-100

Quit Conto asss

Endonuclease Activity: Inubation o a 50 l reation with Wash buer and 1 go X174 RF I DNA or 4 hours at 37c results in < 10% onversion to RF II asdeterined by agarose gel eletrophoresis

Phosphatase Activity: Inubation o a iniu o 10 l o the Wash Buer ata 1X onentration in protein phosphatase assay buer (1 m diethanolaine@ pH 98 and 05 m mgcl

2) ontaining 25 m p-nitrophenyl phosphate at

37c or 4 hours yields no detetable p-nitrophenylene anion as deterined byspetrophotoetri analysis at 405 n

16-Hour Incubation: 50 l reations ontaining the Wash Buer at 1Xonentration and 1 g o HindIII digested Labda DNA inubated or 16hours at 37c results in no detetable non-speif nulease degradation asdeterined by agarose gel eletrophoresis 50 l reations ontaining thisreation buer at 1X onentration and 1 g T3 DNA inubated or 16 hoursat 37c also results in no detetable non-speif nulease degradation asdeterined by agarose gel eletrophoresis

RNase Activity: Inubation o the Wash Buer at a 1X onentration with 40 ngo a FAm-labeled RNA transript or 16 hours at 37c results in no detetableRNase Ativity as deterined by polyarylaide gel eletrophoresis

Lot Controlled

NEbNext Eution buffe#E7494A: 6.0 ml#E7494AA: 24.0 ml

Store at 4C

1X NEBNext Elution Buffer:


Quit Conto asss

Endonuclease Activity: Inubation o a 50 l reation with Elution buer and1 g o X174 RF I DNA or 4 hours at 37c results in less than 10% onversionto RF II as deterined by agarose gel eletrophoresis

Phosphatase Activity: Inubation o a iniu o 10 l o the Elution Buerat a 1X onentration in protein phosphatase assay buer (1 m diethanolaine@ pH 98 and 05 m mgcl



16-Hour Incubation: 50 l reations ontaining the Elution Buer at 1Xonentration and 1 g o HindIII digested Labda DNA inubated or 16hours at 37c results in no detetable non-speif nulease degradation asdeterined by agarose gel eletrophoresis 50 l reations ontaining theElution buer at 1X onentration and 1 g T3 DNA inubated or 16 hoursat 37c also results in no detetable non-speif nulease degradation asdeterined by agarose gel eletrophoresis

RNase Activity: Inubation o the Elution Buer at a 1X onentration with40 ng o a FAm-labeled RNA transript or 16 hours at 37c results in nodetetable RNase Ativity as deterined by polyarylaide gel eletrophoresis

Lot Controlled


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Nucese-fee Wte#E7495A: 1.2 ml#E7495AA: 4.8 ml

Store at 20C or 4C

Description: Nulease-ree Water is ree o detetable DNA and RNA nuleasesand phosphatases and suitable or use in DNA and RNA appliations

Quit Conto asss

16-Hour Incubation: 50 l reations ontaining Nulease-ree Water and 1 go HindIII digested Labda DNA inubated or 16 hours at 37c results in nodetetable non-speif nulease degradation as deterined by agarose gel

eletrophoresis 50 l reations ontaining Nulease-ree Water and 1 g oT3 DNA inubated or 16 hours at 37c results in no detetable non-speifnulease degradation as deterined by agarose gel eletrophoresis

Endonuclease Activity: Inubation o a 50 l reation ontaining Nulease-ree Water with 1 g o X174 RF I DNA or 4 hours at 37c results in < 10%onversion to RF II as deterined by gel eletrophoresis

RNase Activity: Inubation o a 10 l reation ontaining Nulease-ree Waterwith 40 ng o a FAm-labeled RNA transript or 16 hours at 37c results in nodetetable RNase ativity as deterined by polyarylaide gel eletrophoresis

Phosphatase Activity: Inubation o a iniu o 10 l o nulease ree waterat a 1X onentration in protein phosphatase assay buer (1 m diethanolaine@ pH 98 and 05 m mgcl



Lot Controlled


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Vesion 1.18/12

DNA CloNiNg

DNA AmplifiCAtioN & pCR

EpigENEtiCS

RNA ANAlySiS

liBRARy pREp foR NExt gEN SEquENCiNg

pRotEiN ExpRESSioN & ANAlySiS

CEllulAR ANAlySiS

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Teephone: 010-82378265/82378266Fx: 010-82378262e-mi: [email protected]

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