EndoFree Plasmid ezFlow Midi Kit

Introduction

Key to the kit is our proprietary DNA binding systems that DNA exclusively and efficiently binds to our ezBind^TM matrix while proteins and other impurities are removed by wash buffer. Nucleic acids are easily eluted with sterile water or Elution Buffer. Unlike other kits in the markets, no chaotropic salts are contained in the buffer of our patented plasmid purification kit. The purified DNA is guanidine/anion exchange resin residues free.

Plasmid isolated with traditional protocol normally contains high level of endotoxins (Lipopolysaccharides or LPS). For transfection of endotoxin sensitive cell lines or microinjection, the endotoxins should be removed before the applications. The EZgene^TM endofree system uses a specially formulated buffer that extracts the endotoxin from the plasmid DNA. Two rounds of extraction will reduce the endotoxin level to 0.1 EU (Endotoxin) per µg of plasmid DNA. The endofree plasmid miniprep kit provides an efficient endotoxin removal step into the traditional purification procedure to produce transfection grade plasmid DNA.

This kit is designed for fast and efficient purification of plasmid DNA from 15 to 50 mL of E. coli culture. The mini column has a DNA binding capacity of 250 µg.

The purified endofree DNA is ready for downstream applications such as transfection of endotoxin-sensitive cell lines, primary cultured cells or microinjection.

Important Notes

Plasmid Copy Numbers: The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid. The protocols are optimized for high copy number plasmid purification. For low copy number plasmids, both the culture volume and the buffer volume need to be scaled up 2 times. Please contact our customer service for further information and reference Table 1 for the commonly used plasmids,

Table 1 Commonly used plasmid.

Host Strains: The strains used for propagating plasmid have significant influence on yield. Host strains such as Top 10 and DH5a yield high-quality plasmid DNA. endA+ strains such as JM101, JM110, HB101, TG1 and their derivatives, normally have low plasmid yield due to either endogenous endonucleases or high carbohydrates released during lysis. We recommend transform plasmid to an endA- strain if the yield is not satisfactory.

Table2 endA strains of E. Coli.

Optimal Cell Mass (OD₆₀₀ x mL of Culture): This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) for 12 -16 hours to a density of OD₆₀₀ 2.0 to 3.0. If rich mediums such as TB or 2xYT are used, make sure the cell density doesn't exceed 3.0 (OD₆₀₀). A high ratio of biomass over lysis buffers result in low DNA yield and purity. The midi column has an optimal biomass of 100-150. For example, if the OD₆₀₀ is 3.0, the optimal culture volume should be 25-50 mL.

Culture Volume: Use a flask or tube 4 times bigger in voulme than the culture medium to secure optimal condition for bacteria growth. Don't exceed the maximum culture volume suggested in the protocol. Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity.

Storage and Stability

Buffer A1 should be stored at 4°C once RNase A is added. All other materials can be stored at room temperature (22-25°C). The Guaranteed shelf life is 12 months from the date of purchase.

Before Starting

Two endotoxin removal procedures are provided. Protocol A removes endotoxin during the purification of plasmid DNA and Protocol B removes endotoxin after the purification of plasmid DNA.

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

Important  

• RNase A: It is stable for more than half a year when stored at room temperature. Spin down the RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use. Store at 4°C.
• Buffer ER should be stored at 4°C.
• Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50°C to dissolve the precipitates before use.
• Keep the cap tightly closed for Buffer B1 after use.
• Make sure the availability of centrifuge, especially, after mixing the lysate with ethanol, the sample needs to be processed immediately by centrifugation.
• Carry out all centrifugations at room temperature.

Materials supplied by users

• 70% ethanol and 100% ethanol.
• High speed centrifuge.
• 30 mL high speed centrifuge tubes.
• 15 mL and 50 mL conical tubes.

Kit Contents

Safety Information

• Buffer N3 contain acetic acid, wear gloves and protective eyewear when handling.
• Buffer N3 and RET contains chaotropic salts, which may form reactive compounds when combines with bleach. Do not add bleach or acidic solutions directly to the preparation waste.

Operating Protocol