−continuedLit. #267

MessageMAX™ T 7 ARCA-CappedMessage Transcription Kit

Cat. No. MMA60710

The MessageMAX™ T7 ARCA-Capped Mes-sage Transcription Kit is optimized to producethe highest yield of RNA having an Anti-ReverseCap Analog (ARCA) (m2

7,3’-OG[5']ppp[5']G) capon the 5'-end. A standard 30 minute, 20 µlMessageMAX reaction yields up to 60 µg ofRNA starting with 1 µg of control template DNA.These yields are made possible by the un-matched properties of the MessageMAX T7 En-zyme Solution which allow it to function in thepresence of high concentrations of NTPs. Theenzyme solution also includes an RNase inhibi-tor to insure the integrity of the RNA product.

Unlike ARCA, conventional m7G[5']ppp[5']G capanalog is asymmetrical and up to 50% of thecapped RNA produced can have the cap in thereverse orientation which produces sub-optimalresults in both in vitro and in vivo translation.1

Since the ARCA molecule included in the Mes-sageMAX T7 ARCA-Capped Message Tran-scription Kit cannot be incorporated in the re-verse orientation, 100% of the caps in the RNAproduced are in the correct orientation, increas-ing the translation efficiency of the RNA.2

The MessageMAX Kit contains a convenientARCA Cap/NTP PreMix in which all four ribonu-cleotides and the cap analog are mixed in a sin-gle solution. The PreMix ensures the optimalconcentration of each NTP and maintains theideal ratio of ARCA to GTP (4:1) to drive the ini-tiation of transcription with an ARCA cap whilestill producing high RNA yields.

Product Specifications

Storage: Store only at –20oC in a freezer with-out a defrost cycle.

Contaminating Activity Assays: All compo-nents of the MessageMAX Kit are free of de-tectable RNase and DNase activities as judgedby agarose gel electrophoresis following over-di-gestion assays, with the exception of the inher-ent nucleolytic function of the DNase I compo-nent.

MessageMAX™ T7 ARCA-CappedMessage Transcription Kit Contents

The MessageMAX™ Kit contains enoughreagents to perform 10 reactions.

MessageMAX™ T7 Enzyme Solution(contains an RNase inhibitor).................. 20 µl

ARCA Cap/NTP PreMix ....................... 80 µl

20 mM GTP......................................... 20 µl

MessageMAX™ 10X Transcription Buffer........................................................... 20 µl

100 mM Dithiothreitol (DTT) ................. 20 µl

RNase-Free Water.................................1 ml

Control Template DNA@ 0.5 µg/µl .........................................2 µg

RNase-Free DNase I ........................... 10 µl

Control Template: The control template is a4.2-kb linearized plasmid, containing a 1.4-kblambda DNA insert, that will produce a 1,375-brunoff transcript when using T7 RNA polymer-ase.

Related Products: The following products arealso available:– MessageMAX™ T7 Capped Message Tran-

scription Kit– AmpliCap-Max™ T7 and T3 High Yield Mes-

sage Maker Kits– AmpliScribe™ T7-Flash™ and T3-Flash™

Transcription Kits– AmpliScribe™ T7, T3, and SP6 High Yield

Transcription Kits– RNA Cap Analogs– RiboScribe™ RNA Probe Synthesis Kits– T7, T3, and SP6 RNA Polymerases– NTP Solutions

MessageMAX, AmpliCap-Max, AmpliCap, AmpliScribe, T7-Flash, T3-Flash, and RiboScribeare trademarks of EPICENTRE, Madison, Wisconsin.

www.EpiBio.com • (800) 284-8474 • (608) 258-3080 • Fax (608) 258-3088

EPICENTRE MessageMAX™ T 7 ARCA-CappedMessage Transcription Kit

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References:

1. Pasquinelli, A.E. et al., (1995) RNA 1, 957.2. Grudzien, E. et al., (2004) RNA 10, 1479.3. Schenborn, E.T. and Mierendorf, R.C. (1985) Nucl. Acids Res. 13, 6223.4. Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), New York, Cold

Spring Harbor Laboratory Press.5. Milligan, J.F. et al., (1987)

Nucl. Acids Res. 15, 8783.6. Hoffman, L.M. and Johnson, M.G. (1994)

BioTechniques 17, 372.

Notes on Using the MessageMAX Kit

1. Template Preparation: Transcription templates should be linear double-stranded DNA with bluntor 5'-protruding ends. Templates containing 3'-protruding ends can produce spurious transcriptsdue to non-specific initiation.3 3'-protruding ends can be readily converted to blunt ends with T4DNA Polymerase.4 PCR products can also be used as MessageMAX templates, provided that theappropriate promoter is present in the amplified sequence or has been incorporated into one ofthe primers used in amplification. Single-stranded DNA can also be used as template, however,the promoter region must be double-stranded. This can be accomplished by annealing a comple-mentary oligonucleotide to the promoter region before transcription.5,6

The quality of the DNA template directly affects the quality of the RNA produced. Generally, DNAis of sufficient quality for use as an MessageMAX transcription template if it is free of contaminat-ing RNase and can be fully digested with restriction enzymes. To confirm that a template is fullylinearized and intact, examine the DNA on an ethidium-stained agarose or polyacrylamide gel priorto use in a MessageMAX reaction.

Templates that give low yields or less than full-length transcripts may contain RNase or other con-taminants. Such templates will usually give better results after the following treatment:4

1) Add Proteinase K to 100-200 mg/ml and SDS to 0.5%.2) Incubate for 30-60 minutes at 37oC.3) Extract with an equal volume of a 1:1 mixture of TE-saturated phenol/chloroform.4) Ethanol precipitate.5) Gently remove the supernatant and rinse the pellet with 70% ethanol.6) Resuspend at 1.0 mg/ml in RNase-Free T10E1 (10 mM Tris-HCl [pH 7.5], 1 mM EDTA).

2. Template Efficiency: Different templates may give different yields, both in terms of total mg perreaction and transcripts per template. The MessageMAX T7 control template typically gives 50-60 mg of RNA. Lower yields from an experimental template could be due to:

a) Quality of template prep: Degraded templates, RNase or contaminants such as phenol,trace metals and SDS may reduce yields.

b) Transcriptional efficiency: Different templates may be transcribed more or less efficientlybased on promoter strength, reinitiation rate and termination efficiency. Yields may also dif-fer based on the overall size of the template or the relative sizes of the insert and vector.

3. Optimizing the Reaction: The recommended reaction conditions are guidelines and should giveexcellent results with most templates. Modifying the protocol may, however, improve results withsome templates. One way to increase yield is to extend the incubation up to three hours (We donot recommend reaction times longer than 3 hours.). A second way to increase yield in somecases is to raise the template concentration. Finally, increasing the reaction temperature from37oC to 42oC may also improve the yield.

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4. Optimizing Yields for Long (>5 kb) Templates: Synthesis of transcripts longer than 5 kb may re-quire the addition of 1-2 µl of 20 mM GTP to the standard protocol on page 3. This may decreasethe percentage of capped transcript (to 50-60%) but it will increase the yield of full length tran-script.

5. Optimizing Yields for Short (<500 b) Templates: Maximizing the yield of short (<500 b) cappedRNA transcripts requires reaction times of 2 hours.

6. Alternative to Ethanol Precipitation: For transcripts ≥100 bases, we recommend purifying theRNA by the simple addition of one volume of 5 M ammonium acetate followed by centrifugation.This method selectively precipitates RNA while leaving DNA, protein and unincorporated NTPs inthe supernatant. The protocol is:

1) Add one volume of 5 M ammonium acetate (20 µl for the typical MessageMAX reaction).2) Incubate for 15 minutes on ice.3) Centrifuge at high speed (~10,000 x g) for 15 minutes at 4oC.4) Wash the pellet in 70% ethanol.5) Resuspend the pellet in RNase-Free H2O or T10E1.

7. Reaction Assembly: Assembly of the reaction at temperatures less than 22oC or in an alternateorder, can result in the formation of an insoluble precipitate. If assembling a master mix for sever-al reactions, the reaction buffer must be diluted with RNase-Free water before the addition ofrNTPs in order to prevent formation of a precipitate. Additionally, storage of the reaction buffer at-70oC may result in the formation of a white precipitate. If this happens, heat the tube at 37oC for5 minutes and mix thoroughly to resuspend the precipitate.

8. Template Concentration: One microgram is the suggested template amount for most transcriptionreactions, however, the optimal amount may vary. If the concentration of the DNA template is lessthan 0.1 µg/µ l, precipitate the DNA and resuspend in the appropriate amount of RNase-Freewater.

9. Molecular Biology Unit: 1 Molecular Biology Unit (MBU) of DNase I digests 1 µg of pUC19 DNAto oligodeoxynucleotides in 10 minutes at 37oC.

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Protocol for Synthesis of Capped RNA

1. Combine the following reaction components at room temperature in the order given:(see Note 7 page 3)

x µl RNase-Free water1 µg linearized template DNA with appropriate promoter2 µl 10X Transcription Buffer8 µl ARCA Cap/NTP PreMix2 µl 100 mM DTT2 µl MessageMAX T7 Enzyme Solution

20 µl Total reaction volume

2. Incubate at 37oC for 30 minutes when making transcripts > 500 bases.Incubate at 37oC for 2 hours when making transcripts < 500 bases.

DNase I Treatment and Ethanol Precipitation

DNase treatment (steps 3-5) can be used to remove the DNA template, but is unnecessary for manyapplications. Ethanol precipitation (step 6) is also optional, but may be desirable if the RNA is to bestored indefinitely.

3. Add 1 µl (1 MBU) of RNase-Free DNase I and incubate for 15 minutes at 37oC.(see Note 9 page 3).

4. Extract with TE-saturated phenol/chloroform, followed by extraction with chloroform.

5. Remove unincorporated NTPs by chromatography (see Sambrook et al., pp. E.34-E.38)4.

6. Ethanol precipitate by adding sodium acetate to 0.3 M, followed by 2.5 volumes of ethanol. Incu-bate at -20oC ≥30 min and collect by centrifugation. Remove the supernatant carefully with a pi-pette and gently rinse the pellet with 70% ethanol (see Note 6 page 3 for alternative protocol).

7. RNA can be stored at –20oC or –70oC as an ethanol pellet or resuspended in RNase-Free water,T10E1 (p. 2) or other suitable buffer.

Anti-reverse cap analogs (ARCAs) are covered by U.S. Patent No. 7,074,596 and other patents issued or pending, licensed or assigned toEPICENTRE. These products are accompanied by a limited non-exclusive license for the purchaser to use the purchased product(s) solelyfor life science research. Purchase of these products does not grant rights to: (1) offer products, components of products or any derivativesthereof for resale; or (2) to distribute or transfer the products, components of products, or any derivatives thereof to third parties. ContactEPICENTRE for information on licenses for uses other than life science research.