Description :

Introduction

The EZgene^TM family of products is an innovative system that radically simplifies the extraction and purification of nucleic acids from a variety of sources. The key to this system is the new ezBind^TM matrix that specifically, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or a low salt buffer.

The EZgene^TM Tissue DNA Kit provides an easy and rapid method for the isolation of genomic DNA for consistent PCR and Southern analysis. Up to 30 mg of animal tissue, culture cells, mouse tail snips and paraffin-embedded tissue can be readily processed at a time. This kit allows for the single or multiple simultaneous processing of samples. There is no need for phenol/chloroform extractions, and time-consuming steps are eliminated (e.g. precipitation using isopropanol or ethanol). Purified DNA can be directly used for most applications such as PCR, Southern Blotting, and Restriction Enzyme Digestion.

Storage and Stability

All EZgene^TM Tissue DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: reconstituted Proteinase K -20°C, and all other materials at RT (22-25°C).

Binding Capacity

Each ezBind^TM DNA mini column can bind approximately 100 µg DNA. Using greater than 30 mg tissue or 1x10⁷ cells is not recommended.

Kit Contents

*Elution Buffer = 10 mM Tris-HCl, pH 8.5

**Add 8 mL (GD2211-00) or 60 mL (GD2211-01) or 96 mL (GD2211-02) absolute ethanol (96-100%) to each DNA Wash Buffer bottle before use.

Safety Information

Buffer BL contains Chaotropic salts. Use gloves and protective eyeware when handling this solution.

Before Starting

EZgene^TM Kits are designed to be simple, fast, and reliable provided that all steps are followed diligently. It is strongly advised that you familiarize yourself with the entire booklet before starting.

Important Notes

• Reconstitute each vial of Protease K 110 μL (GD2211-00), 1.3 mL (GD2211-01), or 5 x 1.3 mL (GD2211-02) of Elution Buffer. Vortex the vial briefly before use. We recommend that you aliquot, and store vials of Proteinase K at -20°C. Bring Proteinase K solution to room temperature before use.
• Dilute DNA Wash Buffer with absolute ethanol as follows:

Add 8 mL (GD2211-00) or 60 mL (GD2211-01) or 96 mL (GD2211-02) absolute ethanol (96-100%) to each bottle.

•  Preheat Elution Buffer to 70°C (in aliquots of 0.5 mL/ sample).
•  For Mouse Tail Protocol have the following ready: Pre-mixed solutions of 220 μL of Buffer BL and 220 μL of absolute ethanol (per sample). Mix by vortexing. They can be prepared fresh, or pre-made and stored at room temperature less than one month.

Materials and Reagents to be supplied by user

•  Tabletop microcentrifuge.
•  Sterile 1.5 mL centrifuge tubes.
•  Shaking water-bath set to 55°C for Tissue Protocol; 65°C for Cultured Cells; 55°C for Mouse Tail.
•  RNase Stock Solution (100 mg/mL) (OPTIONAL).
•  Absolute Ethanol (96-100%).
•  PBS Buffer.

Determination of Yield and Quality

The total DNA yield can be determined by a spectrophotometer using deionized water, Tris-HCl Buffer, or Elution Buffer as a blank. DNA concentration is calculated as:

[DNA] = (Absorbance₂₆₀) x (0.05 µg/µL) x (Dilution Factor)

The quality of DNA can be assessed by measuring absorbance at both 260 nm and 280 nm. An A₂₆₀/A₂₈₀ ratio of 1.7-1.9 corresponds to 85%-95% purity.

Expected yields vary with both amount and type of tissue used.

Typically, 30 mg of fresh tissue will yield 10~40 µg of DNA with two elutions (each 200 µL).

Concentrate DNA

If necessary, the DNA can be concentrated as follows. Add sodium chloride to reach a final concentration of 0.1M followed by 2 x volume of absolute (96-100%) ethanol. Mix well and incubate at -20°C for 10 min. Centrifuge at 10,000 x g for 15 min and discard supernatant. Add 700 μL of 70% ethanol and centrifuge at 10,000 x g for 2 min. Discard supernatant, air dry the pellet for 2 min, and resuspend the DNA in 20 μL of sterile deionized water or 10 mM Tris-HCl, pH 8.5.

Operating Protocol