E.Z.N.A.® Total RNA Kit I
Table of Contents Introduction (2)
Illustrated Protocol (3)
Kit Contents/Storage and Stability (4)
Preparing Reagents (5)
Recommended Settings (6)
Quantification of RNA (7)
Disruption and Homogenization (8)
Animal Cell Protocol (10)
Animal Tissue Protocol (13)
Spin/Vacuum Manifold Protocol (16)
DNase Digestion Protocol (18)
tabletotal函数Troubleshooting Guide (20)
Ordering (22)
Manual Revision:  October 2009
Innovations in nucleic acid isolation
Introduction
The E.Z.N.A.® RNA family of products is an innovative system that radically simplifies the extraction and purification of RNA from a variety of sources. The key to this system
is that it uses the reversible binding properties of the HiBind Matrix in combination
with the speed of mini-column spin technology thereby permitting single or multiple simultaneous processing of samples. There is no need for phenol/chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation, or precipitation with isopropanol or LiCl are eliminated. RNA
purified using the E.Z.N.A.® RNA Purification System is ready for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.
The E.Z.N.A.® Total RNA Kit I can purify up to 100 μg of total RNA from cultured eukaryotic cells or tissue. Normally, 1 x 106 - 1 x 107 eukaryotic cells, or 5-30 mg of tissue, can be processed in a single experiment.  Fresh, frozen or RNALater® stabilized tissues can be used.  Lysis of cells or tissue occurs under denaturing conditions which inactivate RNases. After the homogenization process, samples are applied to the HiBind RNA spin column to which total RNA binds. Cellular debris and other contaminants are effectively washed away after a few quick wash steps. High quality RNA is finally eluted in DEPC treated water. Total RNA greater than 200 nt is isolated using this kit.
For 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 (Product # R6814) as it is specifically designed for effective hemolysis and hemoglobin removal, thereby giving higher RNA yields. Binding Capacity
Each HiBind RNA Mini column can bind approximately 100μg of RNA. Using greater than 30 mg of tissue or 1 x 107 cells is not recommended.
Kit Contents
Storage and Stability
All E.Z.N.A.® Total RNA Kit components are guaranteed for at least 12 months from  the date of purchase when stored at room temperature. During shipment, crystals or precipitation may form in the TRK Lysis Buffer. Dissolve by warming buffer to 37°C.
Preparing Reagents
•  Dilute RNA Wash Buffer II with absolute ethanol (96-100%) as follows:
•  Store diluted RNA Wash Buffer ll at room temperature.
•  Please remember to always wear gloves whenever working with RNA. This will minimize RNase contamination. Use only clean RNase-free disposable plastic pipette tips when us-ing the supplied reagents.
•  Under cool ambient conditions, crystals may form in TRK Lysis Buffer. This is normal; warm at 37°C to dissolve.
•  Optional:  As a preparation step add 20µl of 2-mercaptoethanol (β-mercaptoethanol) per 1 ml of TRK Lysis Buffer in order to achieve a working solution. This mixture can be stored for 1 week at room temperature.

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