Plasmid Midi Kit I

Buffer A1, B1, C1, KB, Elution Buffer, RNase A, ezBindTM Columns (10), User Manual(1)

Introduction

Key to this kit is our proprietary DNA binding systems that allow DNA exclusively and efficiently bind to our ezBindTM 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 buffers, the purified DNA is guanidine/anion exchange resin residues free which enable the high performance of downstream applications.

This kit is designed for fast and efficient purification of plasmid DNA from 15 to 50 mL of E. coli culture. The midi column has a plasmid DNA binding capacity of 250 µg. The yield from 50 mL culture is typically around 150 to 250 µg.

The purified DNA is ready for high performance of 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 times. Please contact our customer service for further information and reference Table1 in Specifications Tab for the commonly used plasmids.

Table 1 Commonly used plasmid and expected yield.

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 PD1712.

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 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. Store 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.
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.
1.5 mL tubes.
Isopropanol if precipitate the plasmid DNA.

Kit Contents

Safety Information

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