Introduction

Key to the kit is our proprietary DNA binding systems that allow 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.

This kit is designed for fast and efficient purification of plasmid DNA from 100 to 250 mL of E. coli culture. The maxi column has a plasmid DNA binding capacity of 1 mg.

The purified plasmid DNA is ready for downstream applications such as transfection of robust cells such as HEK293, restriction mapping, library screening, sequencing, as well as gene therapy and genetic vaccinations.

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 Commonly 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 4 times larger in volume 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

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, and then store at 4^oC.

• Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50^oC to dissolve the precipitates before use.

• Incubating Buffer C1 at 4 ^oC before experiment will decrease the floating precipitates.

• Keep the cap tightly closed for Buffer B1 after use.

• Make sure the availability of centrifuge and vacuum manifold, especially, after mixing the lysate with ethanol, the sample needs to be processed immediately either by centrifugation or vacuum.

• 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

• 50 mL tubes

Kit contents

Safety Information

• Buffer C1 contains acidic acid, wear gloves and protective eyewear when handling.
• Buffer C1 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