Description :

Introduction

The EZgene^TM Mollusks RNA maxi Kit is designed for efficient recovery of total RNA greater than 200 nt from molluscs, roundworms, flatworms, and other invertebrate tissue samples rich in mucopolysaccharides. The procedure relies on the well established properties of the cationic detergent, cetyltrimethyl ammonium bromide (CTAB), in conjunction with the selective RNA binding of silica membrane. Samples are homogenized and lysed in a high salt buffer containing CTAB and extracted with chloroform to remove mucopolysaccharides and denature proteins. Following a rapid alcohol precipitation step, binding conditions are adjusted and RNA further purified using ezBind RNA spin columns. In this way salts, proteins and other contaminants are removed to yield high quality total RNA suitable for downstream applications such as reverse transcription, poly (A)+ mRNA selection, and hybridization techniques.

Storage and Stability

DNase I (optional) should be stored at -20°C. All other components can be stored at room temperature (22-25°C). All kit components are guaranteed for 1 year from the date of purchasing.

Kit Contents

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

Before Starting

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

Important

•  Determine amount of Buffer BML and Buffer LY to be used, add 20 µL β-mercaptoethanol (β-ME) per 1 mL Buffer BML or LY. Buffer BML and Buffer LY contains β-ME can be stored at room temperature for up to 1 month. β-ME is key in denaturing endogenous RNase.
•  Add 8 mL (R6619-00) or 20 mL (R6619-01) or 216 mL (R6619-02) 100% ethanol to RNA Wash Buffer before use. The final concentration of 100% ethanol should be 80%.
•  During shipment or storage under room temperature, crystal may form in certain buffers. Dissolve the precipitates at 37°C before use.
•  Do not freeze the buffers at any time.
•  Preheat the Buffer RB and DEPC-treated ddH₂O (100 µL per sample) at 65°C.
•  Optional: Add 600 µL (R6619-00) or 7.2 mL (R6619-01) or 36 mL (R6619-02) 100% ethanol to DNase Stop Buffer before use.
•  Smaples: fresh tissue is preferred for RNA integrity.

Materials supplied by users

•  Tabletop microcentrifuge.
•  100% ethanol
•  Nuclease-free microfuge tubes
•  β-mercaptoethanol for denaturing the endogenous RNase.
•  Chloroform and isoamyl alcohol (24:1) for removal of polysaccharides and proteins.
•  Optional: DNase I 
   

 Trouble Shooting Guide

Operating Protocol :