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E.Z.N.A.®

Total RNA Kit IIR6934-00R6934-01R6934-025 preps50 preps200 prepsAugust 2011

E.Z.N.A.® Total RNA Kit IITable .3Kit Contents/Storage 6Quantification 7Disruption 8Animal Cell Protocol

.......................................................9Animal Tissue Protocol

.................................................12Spin/Vacuum 15DNase I Digestion Protocol ............................................20Manual Revision: August 2011Innovations in nucleic acid isolation1

IntroductionThe E.Z.N.A.® Total RNA Kit II is designed for isolating total cellular RNA from tissues rich

in fat such as brain adipose tissues. However, this kit can also be used for the isolation of

total RNA from other type of tissues including cultured eukaryotic cells, animal tissues, or

bacteria.

RNA purified using the E.Z.N.A.® Total RNA method is ready for applications such as RT-PCR*, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in

vitro E.Z.N.A.® Total RNA Kit II uses the reversible binding properties of HiBind® matrix,

a new silica-based material. By combining the high lysis efficiency of RNA-Solv Reagent

with OBI’s innovative HiBind® technology, this kit can extract total cellular RNA from all

types of animal or human tissues including fatty tissues such as brain and adipose tissue.

A specifically formulated high salt buffer system allows more than 100 μg RNA molecules

greater than 200 bases to bind to the matrix. Cells or tissues are first homogenized with

RNA-Solv Reagent that inactivates RNases. After adding chloroform, the homogenate is

separated into aqueous and organic phase by centrifugation. The aqueous phase which

contains the RNA is adjusted with ethanol and applied to the HiBind® RNA Mini Column

to which total RNA binds, while cellular debris and other contaminants are washed away.

High-quality RNA is eluted in DEPC isolating total RNA below 200 nt use the miRNA isolation Kit (R7034). For isolating

total RNA from gram-positive bacteria, the recommended kit is the Bacterial RNA Kit

(R6950).While this kit may be used for the isolation of RNA from whole blood, we recommend

that you use the E.Z.N.A.® Blood RNA Kit (R6814) as it is specifically designed for effective

hemolysis and hemoglobin removal, thereby giving higher RNA g CapacityEach HiBind® RNA Mini Column can bind approximately 100 μg RNA. Using greater than

30 mg tissue or 1 x 107 cells is not recommended.2

Centrifugation ProtocolsAdd RNA-Solv Reagent and

LyseHomogenizeAdd ChloroformTransfer Aqueous PhaseAdd EthanolTransfer to a HiBindColumn® RNA

Wash 3xDryEluteVacuum ProtocolAdd RNA-Solv Reagent and

LyseHomogenizeAdd Chloroform

Transfer Aqueous PhaseAdd EthanolApply Sample to ColumnWash 3xDryElute3

Kit ContentsE.Z.N.A.® Total RNA Kit IIProduct NumberPurificationHiBind® RNA Mini Columns2 mL Collection TubesRNA-Solv ReagentRNA Wash Buffer IRNA Wash Buffer IIDEPC WaterInstruction Manual5 PrepsR6934-0055105 x 1 mL5 mL5 mL1.0 mL150 PrepsR6934- x 28 mL50 mL12 mL10 mL1200 PrepsR6934- x 25 mL200 mL50 mL40 mL1Storage and StabilityAll components except RNA-Solv Reagent in Total RNA Kit II should be stored at room

temperature. RNA-Solv Reagent should be store at 2-8 ºC for long term storage. All Total

RNA Kit II components are guaranteed for at least 12 months from the date of purchase

when stored at 22-25°C.4

Preparing ReagentsDilute RNA Wash Buffer II with 100% ethanol as follows and stored at room

R6934-00R6934-01

R6934-02

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