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Tissue RNA Mini Kit

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Tissue RNA Mini Kit
Features :
Tissue,Eukaryotic cells RNA purification miniprep Kit: Purify up to 100μg total RNA from 5×106 Eukaryotic cells or 30mg tissue
Price : $650.00
Quantity :
Catalogue#: R6311-02
  
 

Description :

Introduction

The EZgeneTM Tissue RNA Kit provides an easy and fast method for isolating total RNA from tissues, cultured cells within 30 min. Only trace genomic DNA exists in the purified RNA, which can be eliminated by DNase I treatment (See detail in the protocol) when it is necessary. This kit purifies up to 100 µg of total RNA from eukaryotic cells or animal tissues. The purified RNA is ready for RT-PCR, Northern blotting, polyA+ RNA purification, nuclease protection, and in vitro translation.

Storage and stability

DNase I (optional) should be stored at -20°C. All other components can be stored at room temperature. All kit components are guaranteed for 12 months from the date of purchasing.

Kit contents

Catalog#

R6311-00

R6311-01

R6311-02

Preps

4

50

250

Buffer LY

2.4 mL

28 mL

135 mL

Buffer RB

3 mL

30 mL

135 mL

RNA Wash Buffer

2 mL

20 mL

3 x 24 mL

DEPC-Treated ddH2O

500 µL

10 mL

30 mL

ezBind Columns

4

50

250

Collection Tubes

8

100

500

DNase I (Optional)*

25 u

300 u

1500 u

DNase Stop Buffer

200 µL

2.4 mL

12 mL

*Solutions are not supplied. They could be purchased from Biomiga.

Safety information

Buffer LY contains chaotropic salts, which may form reactive compounds when combines with bleach. Do not add bleach or acidic solutions directly to the preparation waste, wear gloves and protective eyewear when handling.

Before starting

Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps.

Important

  •  Determine the volume of Buffer LY to be used and add 20 µL of b-mercaptoethanol (b-ME) per 1 mL Buffer LY before use. Buffer LY contains b-ME can be stored at room temperature for up to 1 month.
  •  Crystals may form in Buffer LY, dissolve the precipitates at 37 oC before use.
  •  Add 8 mL (R6311-00) or 80 mL (R6311-01) or 96 mL (R6311-02) 100% ethanol to RNA Wash Buffer before use. The final ethanol is 80% (v/v).
  •  Optional: Add 800 µL or 9.6 mL (R6311-01) or 48 mL (R6311-02) 100% ethanol to DNase Stop Buffer before use. The final ethanol is 80% (v/v).

Materials supplied by users

  •  Tabletop microcentrifuge and 1.5 mL sterile tubes.
  •  Vacuum manifold if use vacuum protocol.
  •  100% ethanol.
  •  b-mercaptoethanol.
  •  Optional: DNase I, DNase Buffer

Note: Perform all steps including centrifugation at room temperature

Trouble Shooting Guide

Problems

Possible reasons

Suggested Improvements

Low A260/A280 ratios

Protein contamination

Do a Phenol:Chloroform extraction. Loss of total RNA (up to 40%) should be expected.

Low A260/A280 ratios

Guanidine Thiocyanate contamination

Add 2.5 volumes of ethanol and 0.1M NaCl (final concentration) to precipitate RNA. Incubate for 30 min at -20°C. Centrifuge at 10,000 g for 15 min at 4°C. Resuspend the RNA pellet in DEPC-treated water.

Low Yield

RNA in sample degraded

Freeze samples immediately in liquid nitrogen and store at -70°C after collect it.

Low Yield

The binding capacity of the membrane in the spin column was exceeded

Use of too much tissue sample exceeding the binding capacity of spin column will cause the decreasing of total RNA yield.

Low Yield

Ethanol not added to buffer

Add ethanol to the RNA Wash Buffer and DNase Stop Solution before purification.

Genomic DNA contamination

Too much total RNA sample was used in RT-PCR.

Reduce total RNA amount used in RT-PCR to 50-100 ng.

Genomic DNA contamination

The sample may contain too much genomic DNA.

Reduce the amount of starting tissue in the preparation of the homogenate. Most tissues will not show a genomic DNA contamination problem at 30 mg or less per prep.

Reduce cell numbers to 1-2x105 or increase buffer volume and do multiple loadings to column.

 

Operating Protocol

6311_448