Description : 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 contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or elution buffer.

Unlike other procedures, our patented plasmid purification kit has no guanidine salt in the buffer, the purified DNA is guanidine/ion exchange resin residues free which enable the high performance of downstream applications such as transfection, 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 PD1714.  

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.

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.

Table 3 The optimal cell mass, culture Volume and Binding Capacity for the mega DNA units,

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^oC). 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 steps.

Important:

• RNase A: It is stable for more than half a year when stored at room temperature. Spin down RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use.
• 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.
• The proper volume of buffer ratio of A1:B1:C1: 100% ethanol =1:1:1.2:1.2.
• Make sure the availability of centrifuge and vacuum manifold, especially, after mixing the lysate with ethanol, the sample needs to be processed immediately by vacuum.

Materials supplied by users:

• 70% ethanol and 100% ethanol.
• Pump-driven vacuum system, 500 mL bottle or 1,000 mL bottle (Corning# 430518 or 430282) or equivalent pyrex glass bottles.
• 50 mL conical 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