MEGAclear Kit

Purification for Large Scale Transcription ReactionsPart Number AM1908

A. Product DescriptionThe MEGAclear Purification Kit is designed to purify RNAfrom enzymatic reactions such as in vitro transcription. Theprocess is simple and fast, and it recovers from 1 ng to 500 gof RNA efficiently. The MEGAclear Kit is appropriate forpurification of ssRNA larger than 200 bp. The MEGAclear Kit procedureis bound to the membra(2) contaminants are washed ain a low salt buffer. The MEGnucleotides, short oligonucleoRNA. The RNA recovered canrequires high purity RNA. Useany of the following: In vitro transcribed RNA:

RNA from MEGAscript Amino allyl-modified RN Biotinyled RNA CyDye labeled RNA Capped RNA (e.g. from

reactions) Total RNAthan 100 nt and dsRNA larger

consists of three steps; (1) RNAne in the Filter Cartridge,way, and (3) the RNA is eluted

Aclear Kit can be used to removetides, proteins, and salts from be used for any application that the MEGAclear Kit to clean up

reactionsA

mMESSAGE mMACHINE

2 B. Kit Content

B. Kit Contents and StorageThe kit contains reagents for 20 RNA purifications.

C. Required Materials Not

100% ethanol: ACS grade or better For preparation of the Was For binding RNA to the Fi

Equipment to pass solutions throug

Microcentrifuge (required) The microcentrifuge mu

10,00015,000 x g (typica

Vacuum manifold (optional) Using a vacuum manifold

vacuum pump) is considerations through the Filter Ca

Use 5 mL syringe barrels tothe vacuum manifold.

Amount Component Storage

8 mL Binding Solution Concentrate 4C

5 mL Wash Solution Concentrate(Add 20 mL 100% ethanol before use)

4C

1 mL 5 M Ammonium Acetate 4C

5 mL Elution Solution any temp*

* Store Elution Solution at room

20 Filter Cartridges room temp

40 Collection and Elution Tubes room temps and Storage

Provided

h Solutionlter Cartridge

h Filter Cartridges

st be capable of attaininglly 10,00014,000 rpm).

(with an adequately powerfulbly faster than drawing the solu-rtridges with a microcentrifuge. support the Filter Cartridges on

temp, 4C, or 20C

D. Buffer and Equipment

D. Buffer and Equipment Preparation

Before Using the Kit for the First Time

Prepare Wash Solution Add 20 mL of ACS grade 100% ethanol to the bottle labeledWash Solution Concentrate. Mix well. Place a check in the boxon the label to indicate that the ethanol was added. With theethanol, this solution will be referred to as Wash Solution.

Equipment Preparation

Lab bench and pipettorsBefore working with RNA, it is always a good idea to clean thelab bench and pipettors with ation (e.g. Ambion RNaseZap

Gloves and RNase-free techWear laboratory gloves at all tchange them frequently. Threagents, and they will protect present on skin.Use RNase-free pipette tips tothe Elution Solution, and avreagent containers.

Microfuge tubesUse the Collection and Elutiothey have been tested for RNafied RNase-free. Preparation 3

n RNase decontamination solu- Solution).

niqueimes during this procedure andey will protect you from thethe RNA from nucleases that are

handle the Wash Solution andoid putting used tips into the

n Tubes supplied with the kit;se contamination and are certi-

4 E. MEGAclear

E. MEGAclear Kit Procedure

CAUTION

Filter Cartridges should not be subjected to RCFs over 16,000 x g becauseit could cause mechanical damage and/or may deposit glass filter fiber inthe final sample.

1. Bring the RNA sample to 100 L with Elution Solution. Mix gently but thoroughly.

2. Add 350 L of Binding Solution Concentrate to the sample. Mix gently by pipetting.

3. Add 250 L of 100% ethanol to thpipetting.

4. Apply the sample to the filter.

Centrifuge users:

a. Insert a Filter Cartridge intTubes supplied.

b. Pipet the RNA mixture ont

c. Centrifuge for ~15 sec to passed through the 10,00015,000 x g (typSpinning harder than this m

d. Discard the flow-throughElution Tube for the washi

Vacuum manifold users:

a. Put 5 mL syringe barrels them with Filter Cartridges

b. Pipet the RNA mixture vacuum will draw it througif the RNA mixture is pullewill bind instantly. Kit Procedure

e sample. Mix gently by

o 1 of the Collection and Elution

o the Filter Cartridge.

1 min, or until the mixture hasfilter. Centrifuge at RCFically 10,00014,000 rpm).ay damage the filters.

and reuse the Collection andng steps.

on the vacuum manifold, load, and apply the vacuum.

onto the Filter Cartridge. Theh the filter. Do not be concernedd through very quickly, the RNA

E. MEGAclear Kit P

5. Wash with 2 x 500 L Wash SolutionMake sure that the ethanol has been added to the Wash Solu-tion Concentrate before using it.

a. Apply 500 L Wash Solution. Draw the Wash Solutionthrough the filter as in the previous step.

b. Repeat with a second 500 L aliquot of Wash Solution.

c. After discarding the Wash Solution, continue centrifugationor leave the Filter Cartridge on the vacuum manifold for1030 sec to remove the last traces of Wash Solution.

6. Elute RNA with 50 L Elution SolutionElute the RNA from the filter using either one of the methodsdescribed below; they are equiv

RNA elution option 1

a. Place the Filter Cartridge int

b. Apply 50 L of Elution SoCartridge. Close the cap ofblock set to 6570C for 5

c. Recover eluted RNA by cen10,00015,000 x g).

d. To maximize RNA recovewith a second 50 L aliquoeluate into the same tube.

RNA elution option 2

a. Pre-heat 110 L of Elution

b. Apply 50 L of the pre-heatof the Filter Cartridge, ccentrifuge for 1 min a10,00015,000 x g) to elut

c. To maximize RNA recovewith a second pre-heated 50Collect the eluate into the s

NOTE

If glass fibers are observed in your sacentrifuging the sample briefly and then trrocedure 5

alent in terms of RNA recovery.

o a new Collection/Elution Tube.

lution to the center of the Filter the tube and incubate in a heat10 min.

trifuging for 1 min at RT (RCF

ry, repeat this elution proceduret of Elution Solution. Collect the

Solution per sample to 95C.

ed Elution Solution to the centerlose the cap of the tube andt room temperature (RCF

e the RNA.

ry, repeat this elution procedureL aliquot of Elution Solution.

ame Collection/Elution Tube.

mple, they can be removed byansferring the RNA to a new tube.

6 F. Assessing

7. (optional) Precipitate with 5 M Ammonium AcetateTo concentrate the RNA, precipitate as follows:

a. Add 1:10 volume of 5 M Ammonium Acetate (NH4Ac) tothe purified RNA. If the sample was eluted with 100 L Elution Solution assuggested, this will be 10 L of 5 M NH4Ac.

b. Add 2.5 volumes of 100% ethanol (275 L if the RNA waseluted in 100 L). Mix well and incubate at 20C for30 min.

c. Microcentrifuge at top speed for 15 min at 4C or roomtemperature (RT).

d. Carefully remove and discard the supernatant.

e. Wash the pellet with 500 again and remove the 70%

f. To remove the last traces tube, and aspirate any residpipette, or with a syringe n

g. Air dry the pellet.

h. Resuspend the pellet using

F. Assessing RNA Yield

1. Assessing RNA yield by UV absoThe concentration of RNA caaliquot of the preparation (usuTE (10 mM Tris-HCl pH 8, absorbance in a spectrophotomfor dilution need not be RNasethe RNA), since slight degradaicantly affect its absorbance. tometer with the TE used for An A260 of 1 is equivalent to 4The concentration (g/mL) ofollows: A260 X dilution factor RNA Yield

L 70% cold ethanol, centrifugeethanol.

of ethanol, quickly re-spin theual fluid with a very fine tipped

eedle.

the desired solution and volume.

rbancen be determined by diluting anally a 1:50 to 1:100 dilution) in1 mM EDTA), and reading theeter at 260 nm. The buffer used-free (unless you want to recovertion of the RNA will not signif-

Be sure to zero the spectropho-sample dilution.0 g RNA/mL. f RNA is therefore calculated asX 40 g/mL.

G. Quality Cont

2. Assessing RNA yield with Ribogreen

Ribogreen (Invitrogen) provides a sensitive method for quanti-tating RNA in solution. Follow the manufacturers instructionsfor use.

G. Quality Control

Functional TestingAn entire MEGAscript reaction is purified, and recovery isshown to be >75% of input RNA. The RNA is then reversetranscribed using a trace radiolabel and the reaction productsare analyzed by PAGE.

Nuclease testingRelevant kit components are assays:

RNase activityMeets or exceeds specification labeled RNA and analyzed by

Nonspecific endonuclease aMeets or exceeds specification supercoiled plasmid DNA andphoresis.

Exonuclease activityMeets or exceeds specification labeled double-stranded DNArol 7

tested in the following nuclease

when a sample is incubated withPAGE.

ctivitywhen a sample is incubated with analyzed by agarose gel electro-

when a sample is incubated with, followed by PAGE analysis.

8 H. Safety In

H. Safety Information

Chemical safety guidelinesTo minimize the hazards of chemicals: Read and understand the Material Safety Data Sheets

(MSDS) provided by the chemical manufacturer before youstore, handle, or work with any chemicals or hazardousmaterials.

Minimize contact with chemicals. Wear appropriate per-sonal protective equipment when handling chemicals (forexample, safety glasses, gloves, or protective clothing). Foradditional safety guidelines, consult the MSDS.

Minimize the inhalation ofcal containers open. Use onexample, fume hood). For sult the MSDS.

Check regularly for chemicoccurs, follow the manufrecommended on the MSD

Comply with all local, stateregulations related to chemposal.

About MSDSsChemical manufacturers suppSheets (MSDSs) with shipmencustomers. They also provide of a hazardous chemical to a cuupdated. MSDSs provide thestore, handle, transport, and dEach time you receive a new Mchemical, be sure to replace files.

Obtaining the MSDSTo obtain Material Safety Datical product supplied by Appliformation

chemicals. Do not leave chemi-ly with adequate ventilation (for

additional safety guidelines, con-

al leaks or spills. If a leak or spillacturers cleanup procedures asS./provincial, or national laws andical storage, handling, and dis-

ly current Material Safety Datats of hazardous chemicals to newMSDSs with the first shipmentstomer after an MSDS has been

safety information you need toispose of the chemicals safely.SDS packaged with a hazardous

the appropriate MSDS in your

a Sheets (MSDSs) for any chem-ed Biosystems or Ambion:

H. Safety Informa

At www.appliedbiosystems.com, select Support, thenMSDS. Search by chemical name, product name, productpart number, or MSDS part number. Right-click to print ordownload the MSDS of interest.

At www.ambion.com, go to the web catalog page for theproduct of interest. Click MSDS, then right-click to printor download.

E-mail (MSDS_Inquiry_CCRM@appliedbiosystems.com)or telephone (650-554-2756; USA) your request, specifyingthe catalog or part number(s) and the name of the prod-uct(s). We will e-mail the associated MSDSs unless yourequest fax or postal delivery. Requests for postal deliveryrequire 12 weeks for processing.

For the MSDSs of chemicals ntems or Ambion, contact the ction 9

ot distributed by Applied Biosys-hemical manufacturer.

10 H. Safety Information

H. Safety Informa

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Manual 1908M Revision B Revision Date: August 8, 2008

For research use only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice. Applied Biosystemsassumes no responsibility for any errors that may appear in this document.Applied Biosystems disclaims all warranties with respect to this document, expressed orimplied, including but not limited to those of merchantability or fitness for a particularpurpose. In no event shall Applied Biosystems be liable, whether in contract, tort, war-ranty, or under any statute or on any other basis for special, incidental, indirect, puni-tive, multiple or consequential damages in connection with or arising from thisdocument, including but not limited to the use thereof.Literature Citation: When you are describing a procedure utilizing this product in aMaterials and Methods Section for publication, we would appreciate that you refer to itas the MEGAclear Kit.Warranty and Liability: Applied Biosystems is committed to delivering superior prod-uct quality and performance, supported by industry-leading global service and technicalsupport teams. Warranty information for the accompanying consumable product isavailable at www.ambion.com/info/warranty in Limited Warranty for Consumables,which is subject to the exclusions, conditions, exceptions, and limitations set forth underthe caption EXCLUSIONS, CONDITIONS, EXCEPTIONS, AND LIMITA-TIONS in the full warranty statement. Please contact Applied Biosystems if you haveany questions about our warranties or would like information about post-warranty sup-port.Trademarks: Applied Biosystems, AB (Design), Ambion, MEGAscript, mMESSAGEmMACHINE, and RNaseZap are registered trademarks; and MEGAclear is a trademarkof Applied Biosystems Inc. or its subsidiaries in the US and/or certain other countries. All other trademarks are the sole property of their respective owners.

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12

A. Product DescriptionB. Kit Contents and StorageC. Required Materials Not Provided100% ethanol: ACS grade or betterEquipment to pass solutions through Filter Cartridges

D. Buffer and Equipment PreparationBefore Using the Kit for the First TimeEquipment Preparation

E. MEGAclear Kit Procedure1. Bring the RNA sample to 100 L with Elution Solution. Mix gently but thoroughly.2. Add 350 L of Binding Solution Concentrate to the sample. Mix gently by pipetting.3. Add 250 L of 100% ethanol to the sample. Mix gently by pipetting.4. Apply the sample to the filter.5. Wash with 2 x 500 L Wash Solution6. Elute RNA with 50 L Elution Solution7. (optional) Precipitate with 5 M Ammonium Acetate

F. Assessing RNA Yield1. Assessing RNA yield by UV absorbance2. Assessing RNA yield with Ribogreen

G. Quality ControlFunctional TestingNuclease testing

H. Safety InformationChemical safety guidelinesAbout MSDSsObtaining the MSDSSend Us Your Reprint & Well Give You The Shirt Off Our Back!

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