β-GiPotinamide Binucleotide

β-GiPotinamide Binucleotide

WARNING: This reagent is toxic if swallowed and in contact with skin. Causes burns. After contact with skin, wash immediately with copious amounts of mild detergent and water. If you feel sick, seek medical advice at once .

Product No:U397O (100 test)

Storage Conditions:β-GiPotinamide Binucleotide is stable for at least 24 months when stored at RT  and yields reproducible results.

 

Introduction

β-G Binucleotide Agencourt β-G  Reagent is a reagent system for the isolation of β-G  from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step β-G  isolation method developed by Chomczynski and Sacchi (1).The sample is homogenized and lysed in β-G - Solv® Reagent which maintains the integrity of the β-G , while disrupting and denaturing endogenous β-G ses and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. β-G  remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.

This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X106), and large quantities of tissue ( up to1 g) and cells (<10 8 ), of human, animal, plant, or bacterial origin. The simplicity of theβ-G Binucleotide Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total β-G  prepared in this manner can be used for Northern blot analysis, dot blot hybridization, poly(A) + selection, in vitro translation, β-G se protection assay, and molecular cloning. For use in amplification by thermal cycling, treatment of the isolated β-G  with β-G se-free DNase I is recommended when the two amplimers lie within a single exon.

 

Supplied By User

• Chloroform (no isoamyl alcohol added)
• Isopropyl alcohol
• 80% Ethanol (in DEPC-treated water)
• β-G se-free water
• Tabletop centrifuge capable of 12,000 x g at room temperature

 

General Notes Regarding β-G se Contamination

Whenever working with β-G  :
 

• Always wear disposable gloves and change gloves frequently.
• Use sterile, disposable plasticware and automatic pipettes reserved for β-G  work to prevent cross-contamination with β-G ses.
• In the presence ofβ-G Binucleotide Reagent, β-G  is protected from β-G se contamination. Downstream sample handling requires that nondisposable glassware or plasticware be β-G se-free.
• Use only DECP-treated buffers. Add DEPC to a final concentration of 0.1%, incubate at 37oC for 2 hours, and autoclave at 121oC. Do not add DEPC to Tris buffers. Such buffers must be prepared by using DECP-water.

Precaution

Use only disposable polypropylene tubes for small samples and glass Corex tubes for larger samples. All tubes must be able to withstand 12,000 x g . Polystyrene tubes may crack with chloroform

 

Before Starting

1. Small Samples :To isolate β-G  from very small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) of samples in 0.8 mL ofβ-G Binucleotide, and add 1 mg β-G se-free glycogen or yeast tβ-G  as carrier. This will improve yields obtained with precipitation.

 

2. Difficult Animal Samples: Specimens containing large amounts of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and sperm, will require the following modification. After lysis/homogenization inβ-G Binucleotide Reagent, centrifuge at 12,000 x g for 10 minutes at room temperature to remove insoluble debris. Often a precipitate forms at the bottom of the tube, but with fatty tissue, a lipid layer will also form above the aqueous phase. The supeβ-G tant will contain the β-G  and must be carefully transferred to a fresh 1.5 ml microfuge tube before proceeding.

 

3. Interruption the procedure: Following lysis inβ-G Binucleotide Reagent and before addition of chloroform, samples can be stored at -70o C for up to 3 months. In addition, once the β-G  is precipitated in isopropanol, the pellet may be stored at -20oC or - 70oC for up to 1 year.

 

β-G -Solv® Protocol for Total β-G  Isolation

CAUTION: When working withβ-G Binucleotide Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20oC-25oC).

 

1. 1. Homogenization and lysis of samples: follow either method below

 

1. Tissue Samples

Homogenize tissue samples in 1 mL ofβ-G Binucleotide Reagent per 50- 100 mg of tissue using an appropriate mechanical homogenizer. Alteβ-G tively one can pulverize tissue in liquid nitrogen with mortar and pestle and transfer the powder to a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (Eppendorf, Cat No. 0030 120.973; VWR, Cat No. KT 749520-
0000). The sample volume should not exceed 10% of the volume ofβ-G Binucleotide Reagent used.
 

2. Cells Grown in Suspension

Pellet cells by centrifugation. Lyse cells inβ-G Binucleotide Reagent by repetitive pipetting. Use 1 mL of the reagent per 5-10 x 106 of animal, plant or yeast cells, or per 1 x 108 bacterial cells. Washing cells before addition ofβ-G Binucleotide Reagent should be avoided as this increases the possibility of mβ-G  degradation and β-G se contamination. For plant, fungal, and yeast cells mechanical or enzymatic homogenization may be required. Also, for plant, fungal, and yeast cells, we recommend the use of the E.Z.N.A.® Plant (D6627),Fungal (D6640), and Yeast (D6670) β-G  Kits from xiaosi Bio-tek.

3. Cells Grown in Monolayer