Description :

Introduction

The EZgeneTM high-capacity-plasmid DNA Isolation Kit is designed for rapid purification of BAC, PAC, cosmid and P1 from bacterial cultures. It is based on a modified alkaline lysis procedure that is specially adapted for spin column. The procedure associated with this kit has been tested using a variety of low copy cosmid, BAC, PAC and P1 in different E. coli strains. In addition, this kit can also be used for high copy plasmid isolation. The expected yield from 200 mL of 2 x YT is around 20 to 80 μg.

Storage and Stability

All EZgeneTM BAC/PAC DNA Isolation Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: Buffer A1/RNase A mixture at 4°C, and all other materials at 22-25°C.

Kit Contents

* Binding Buffer: Add 24 mL (PD1314-00) or 100 mL (PD1314-01) or 280 mL (PD1314-02) of isopropanol before use. **DNA Wash Buffer: Add 24 mL (PD1314-00) or 100 mL (PD1314-01) or 216 mL (PD1314-02) of absolute ethanol before use.

Before Starting

Important

 Add whole vial of RNase A to Buffer X1 and Store at 4°C.

 Dilute DNA Wash Buffer with absolute ethanol as follows

 PD1314-00 Add 20 mL ethanol to each bottle

 PD1314-01 Add 100 mL ethanol to each bottle

 PD1314-02 Add 240 mL ethanol to each bottle

 It's strongly recommended to use 2 x YT media for the cultivation of cosmids, BACs, PACs, and P1s.

 Dilute BAC Binding Buffer with isopropanol

PD1314-00 Add 24 mL isopropanol to each bottle PD1314-01 Add 120 mL isopropanol to each bottle PD1314-02 Add 280 mL isopropanol to each bottle

 Buffer X2 should be kept at room temperature. Check for SDS precipitation before use. If necessary re-dissolve SDS precipitate by warming. Close the Buffer X2 bottle immediately after use to avoid acidification that may result from air CO2.

 Chill Buffer X3 on ice.

 Prewarm ddH2O or Elution Buffer at 65°C before elution

Materials Supplied by User

 High speed centrifuge and centrifuge tubes capable of at least 12,000 x g

 Sterile Deionized Water

 50 mL conical tubes.

 96-100% Ethanol

 96%-100% Isopropanol

 One liter 2 x YT medium: Measure 900 mL ddH2O, add 16 g Tryptone, 10 g Yeast Extract, 5 g NaCl, dissolve and adjust pH to 7.0 with 5 N NaOH, adjust to 1000 mL with ddH2O and autoclave.