Description :

Introduction

Ezgene^TM Fungal RNA Miniprep Kit provides a rapid and reliable method for isolation of total RNA from a wide variety of fungal samples. The kit does not require  the  use  of  cumbersome  or  expensive  shredding/homogenizing accessories  as  an attempt to shear viscous  fungal  lysates.   Rather, then method involves a simple and rapid precipitation step for removal of much of the polysachrides and phenolic compounds commonly found in fungal tissues. In combination with ezBind ^TM RNA spin columns, this method permits purification of high quality RNA from as much as 200 mg tissue. The system is efficient enough to allow total RNA from as little as 10 mg of tissue or 100 cells. Typical yields are shown in Table 1. The procedure involves no organic extractions, thus reducing plastic waste and hands-on time.    Ezgene ^TM Fungal RNA Kits are ideal for processing multiple fungal samples in parallel in 1 hour. Purified RNA has A₂₆₀/A₂₈₀ ratios of 1.8-20 and is suitable for RT-PCR, Northern Analysis,        Differential display, Poly A+ RNA selection.

Storage and Stability

All components of the Ezgene^TM Fungal RNA Kit are stable for at least 12 months from date of purchase when stored at 22^oC-25^oC.

Binding Capacity

Each ezBind^TM RNA column can bind approximately 100 µg RNA. Using greater than 250 mg fungal tissue in many cases will not drama tical improve yields and sometimes has adverse affects.

Kit Contents

Note：

Buffer FLY contains a chaotropic salt. Use gloves and protective eyeware when handling this solution.

DNase I are not supplied. They could be purchased from Biomiga.

Before Starting

Important

 Please read the entire booklet to become familiar with the Ezgene^TM Fungal RNA procedure.

Dilute RNA Wash Buffer Concentrate with ethanol as follows:

Add 8 mL (R6618-00), 80 mL(R6618-01), 96 mL(R6618-02) absolute (96%-100%) ethanol to  each bottle before use.

Add 20 µL ß-mercaptoethanol per 1 mL of Buffer FLY.

Materials to be provided by user

•  Microcentrifuge capable of 10,000 x g
•  Nuclease-free microfuge tubes
•  ß -mercaptoethanol
•  Absolute (96%-100%) ethanol
•  Liquid nitrogen for freezing/disrupting samples
•  Preheat an aliquot (100 µL per sample) of DEPC-treated water at 65^oC.

Working with RNA

Please take a few minutes to read this booklet thoroughly to become familiar with the protocol. Prepare all materials required before starting to minimize RNA degradation.

•  Whenever working with RNA, always wear latex gloves to minimize RNase contamination. Change gloves frequently. Use only clean RNase-free disposable plastic pipette tips when using the supplied reagents.
•  During the procedure work carefully but quickly.
•  During the procedure work carefully but quickly.
•  ß-mercaptoethanol (ß-mercaptoethanol) is key in denaturing endogenous RNases and must be added to an aliquot of Buffer FLY before use. Add 20 µL of ß-mercaptoethanol per 1 ml of Buffer FLY. This mixture can be stored for 1 week at room temperature.

RNA Quality

It is highly recommended that RNA quality be determined prior to all analyses. The quality of RNA can be assessed  by denaturing agarose gel electrophoresis and ethidium bromide staining.  Several sharp bands should appear on the gel. These are the 28S and 18S ribosomal RNA bands as well as certain populations of mRNA and possibly viral RNA bands. If these bands smear towards lower molecular weight RNAs, then the RNA has undergone major degradation during preparation, handling, or storage. RNA molecules less than 200 bases in length do not efficiently bind the ezBind matrix, thus the method enriches high quality RNA. Since no RNA extraction procedure can completely remove genomic DNA. For sensitive work (such as RT-PCR or differential display) either on-membrane DNase I digestion treatment or after elution DNase I digestion will be needed. For modified protocols for DNase I digestion, call our technical staff.

Trouble Shooting Guide

Operating Protocol :