MEGAclear™ Kit

Purification for Large Scale Transcription ReactionsPart Number AM1908

A. Product Description

The 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 μgof RNA efficiently. The MEGAclear Kit is appropriate forpurification of ssRNA larger than 100 nt and dsRNA largerthan 200 bp. The MEGAclear Kit procedure consists of three steps; (1) RNAis bound to the membrane in the Filter Cartridge,(2) contaminants are washed away, and (3) the RNA is elutedin a low salt buffer. The MEGAclear Kit can be used to removenucleotides, short oligonucleotides, proteins, and salts fromRNA. The RNA recovered can be used for any application thatrequires high purity RNA. Use the MEGAclear Kit to clean upany of the following:• In vitro transcribed RNA:

– RNA from MEGAscript® reactions– Amino allyl-modified RNA– Biotinyled RNA– Cy™Dye labeled RNA– Capped RNA (e.g. from mMESSAGE mMACHINE®

reactions)• Total RNA

B. Kit Contents and Storage

The kit contains reagents for 20 RNA purifications.

C. Required Materials Not Provided

100% ethanol: ACS grade or better

• For preparation of the Wash Solution• For binding RNA to the Filter Cartridge

Equipment to pass solutions through Filter Cartridges

Microcentrifuge (required)

• The microcentrifuge must be capable of attaining10,000–15,000 x g (typically 10,000–14,000 rpm).

Vacuum manifold (optional)

• Using a vacuum manifold (with an adequately powerfulvacuum pump) is considerably faster than drawing the solu-tions through the Filter Cartridges with a microcentrifuge.

• Use 5 mL syringe barrels to support the Filter Cartridges onthe vacuum manifold.

Amount Component Storage

8 mL Binding Solution Concentrate 4°C

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

4°C

1 mL 5 M Ammonium Acetate 4°C

5 mL Elution Solution any temp*

* Store Elution Solution at room temp, 4°C, or –20°C

20 Filter Cartridges room temp

40 Collection and Elution Tubes room temp

2 B. Kit Contents and Storage

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 pipettors

Before working with RNA, it is always a good idea to clean thelab bench and pipettors with an RNase decontamination solu-tion (e.g. Ambion® RNaseZap® Solution).

Gloves and RNase-free technique

Wear laboratory gloves at all times during this procedure andchange them frequently. They will protect you from thereagents, and they will protect the RNA from nucleases that arepresent on skin.Use RNase-free pipette tips to handle the Wash Solution andthe Elution Solution, and avoid putting used tips into thereagent containers.

Microfuge tubes

Use the Collection and Elution Tubes supplied with the kit;they have been tested for RNase contamination and are certi-fied RNase-free.

D. Buffer and Equipment Preparation 3

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 μL with Elution Solution. Mix

gently but thoroughly.

2. Add 350 μL of Binding Solution Concentrate to the sample.

Mix gently by pipetting.

3. Add 250 μL of 100% ethanol to the sample. Mix gently by

pipetting.

4. Apply the sample to the filter.

Centrifuge users:

a. Insert a Filter Cartridge into 1 of the Collection and ElutionTubes supplied.

b. Pipet the RNA mixture onto the Filter Cartridge.

c. Centrifuge for ~15 sec to 1 min, or until the mixture haspassed through the filter. Centrifuge at RCF10,000–15,000 x g (typically 10,000–14,000 rpm).Spinning harder than this may damage the filters.

d. Discard the flow-through and reuse the Collection andElution Tube for the washing steps.

Vacuum manifold users:

a. Put 5 mL syringe barrels on the vacuum manifold, loadthem with Filter Cartridges, and apply the vacuum.

b. Pipet the RNA mixture onto the Filter Cartridge. Thevacuum will draw it through the filter. Do not be concernedif the RNA mixture is pulled through very quickly, the RNAwill bind instantly.

4 E. MEGAclear™ Kit Procedure

5. Wash with 2 x 500 μL Wash Solution

Make sure that the ethanol has been added to the Wash Solu-tion Concentrate before using it.

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

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

c. After discarding the Wash Solution, continue centrifugationor leave the Filter Cartridge on the vacuum manifold for10–30 sec to remove the last traces of Wash Solution.

6. Elute RNA with 50 μL Elution Solution

Elute the RNA from the filter using either one of the methodsdescribed below; they are equivalent in terms of RNA recovery.

RNA elution option 1

a. Place the Filter Cartridge into a new Collection/Elution Tube.

b. Apply 50 μL of Elution Solution to the center of the FilterCartridge. Close the cap of the tube and incubate in a heatblock set to 65–70°C for 5–10 min.

c. Recover eluted RNA by centrifuging for 1 min at RT (RCF10,000–15,000 x g).

d. To maximize RNA recovery, repeat this elution procedurewith a second 50 μL aliquot of Elution Solution. Collect theeluate into the same tube.

RNA elution option 2

a. Pre-heat 110 μL of Elution Solution per sample to 95°C.

b. Apply 50 μL of the pre-heated Elution Solution to the centerof the Filter Cartridge, close the cap of the tube andcentrifuge for 1 min at room temperature (RCF10,000–15,000 x g) to elute the RNA.

c. To maximize RNA recovery, repeat this elution procedurewith a second pre-heated 50 μL aliquot of Elution Solution.Collect the eluate into the same Collection/Elution Tube.

NOTE

If glass fibers are observed in your sample, they can be removed bycentrifuging the sample briefly and then transferring the RNA to a new tube.

E. MEGAclear™ Kit Procedure 5

7. (optional) Precipitate with 5 M Ammonium Acetate

To 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 μL Elution Solution assuggested, this will be 10 μL of 5 M NH4Ac.

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

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

d. Carefully remove and discard the supernatant.

e. Wash the pellet with 500 μL 70% cold ethanol, centrifugeagain and remove the 70% ethanol.

f. To remove the last traces of ethanol, quickly re-spin thetube, and aspirate any residual fluid with a very fine tippedpipette, or with a syringe needle.

g. Air dry the pellet.

h. Resuspend the pellet using the desired solution and volume.

F. Assessing RNA Yield

1. Assessing RNA yield by UV absorbance

The concentration of RNA can be determined by diluting analiquot of the preparation (usually a 1:50 to 1:100 dilution) inTE (10 mM Tris-HCl pH 8, 1 mM EDTA), and reading theabsorbance in a spectrophotometer at 260 nm. The buffer usedfor dilution need not be RNase-free (unless you want to recoverthe RNA), since slight degradation of the RNA will not signif-icantly affect its absorbance. Be sure to zero the spectropho-tometer with the TE used for sample dilution.An A260 of 1 is equivalent to 40 μg RNA/mL. The concentration (μg/mL) of RNA is therefore calculated asfollows: A260 X dilution factor X 40 μg/mL.

6 F. Assessing RNA Yield

2. Assessing RNA yield with Ribogreen®

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

G. Quality Control

Functional Testing

An 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 testing

Relevant kit components are tested in the following nucleaseassays:

RNase activity

Meets or exceeds specification when a sample is incubated withlabeled RNA and analyzed by PAGE.

Nonspecific endonuclease activity

Meets or exceeds specification when a sample is incubated withsupercoiled plasmid DNA and analyzed by agarose gel electro-phoresis.

Exonuclease activity

Meets or exceeds specification when a sample is incubated withlabeled double-stranded DNA, followed by PAGE analysis.

G. Quality Control 7

H. Safety Information

Chemical safety guidelines

To 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 of chemicals. Do not leave chemi-cal containers open. Use only with adequate ventilation (forexample, fume hood). For additional safety guidelines, con-sult the MSDS.

• Check regularly for chemical leaks or spills. If a leak or spilloccurs, follow the manufacturer’s cleanup procedures asrecommended on the MSDS.

• Comply with all local, state/provincial, or national laws andregulations related to chemical storage, handling, and dis-posal.

About MSDSs

Chemical manufacturers supply current Material Safety DataSheets (MSDSs) with shipments of hazardous chemicals to newcustomers. They also provide MSDSs with the first shipmentof a hazardous chemical to a customer after an MSDS has beenupdated. MSDSs provide the safety information you need tostore, handle, transport, and dispose of the chemicals safely.Each time you receive a new MSDS packaged with a hazardouschemical, be sure to replace the appropriate MSDS in yourfiles.

Obtaining the MSDS

To obtain Material Safety Data Sheets (MSDSs) for any chem-ical product supplied by Applied Biosystems or Ambion:

8 H. Safety Information

• 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 1–2 weeks for processing.

For the MSDSs of chemicals not distributed by Applied Biosys-tems or Ambion, contact the chemical manufacturer.

H. Safety Information 9

10 H. Safety Information

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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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