Introduction

Key to the plasmid purification kit is our proprietary DNA binding system that allows the high efficient binding of DNA 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, our patented plasmid purification kit has no chaotropic salts in the buffer. The purified DNA is guanidine/anion exchange resin residues free.

The EZgene^TM endofree system uses a specially formulated buffer that extracts the endotoxin from the bacterial lysate. The endotoxin level is less than 0.1 EU (Endotoxin) per µg of plasmid DNA.

This kit is designed for fast and efficient purification of plasmid DNA from 150 to 400 mL of E. coli culture. The maxi column has a DNA binding capacity of 1200 µg. The purified endofree DNA is ready for downstream applications such as transfection of endotoxin-sensitive cell lines, primary cultured cells or microinjection.

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.

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 to 3 times. Reference Table 1 for the commonly used plasmids,

Table 1 Commomly used plasmids

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. For purifying plasmid DNA from endA+ strains (Table 2), we recommend use product PD1713.

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 medium 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 maxi column has an optimal biomass of 450-550. For example, if the OD₆₀₀ is 2.5, the optimal culture volume should be 200 mL.

Culture Volume: Use a flask or tube with a volume at 4 times 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 and Buffer ER should be stored at 4°C. All other materials can be stored at room temperature (22-25°C). The Guaranteed shelf life is 12 months from the date of purchase.

Kit Contents

Safety Information

Buffer N3, RET contain acetic acid, wear gloves and protective eyewear when handling.

*Buffer ER and RET are for endotoxin removal.

Before Starting

Alternative 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 either by centrifugation.
•  In case the collection tube doesn't fit in high-speed centrifuge rotor, use benchtop centrifuge and spin at 2,500 x g with double centrifugation time. For example, centrifuge at 2,500 x g for 10 min instead of 5,000 x g for 5 min.
•  Carry out all centrifugations at room temperature.

Materials supplied by users

• •  70% ethanol and 100% ethanol
  •  High speed centrifuge or vacuum manifold.
  •  30 mL high speed centrifuge tubes and 50 mL tubes.

  • Operating Protocol