Plasmid midiprep protocol

Key to the kit is the proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBindTM matrix while proteins and other contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or TE buffer. Unlike all other rivals, 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

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 3 to 5 times. Please contact our customer service for further information and reference the table below for the commonly used plasmids.

Storage and Stability

Buffer A1 should be stored at 4℃ once RNase A is added. All other materials can be stored at room temperature. The Guaranteed shelf life is 18 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:

R RNase A: Spin down RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use.

R Buffer B1 precipitates below room temperature, it is critical to warm up the buffer at 37℃ to dissolve the precipitates before use.

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

R 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.

R Carry out all centrifugations at room temperature.

EZgeneTM Plasmid Midiprep Spin Protocol

1. Inoculate 50-80 mL LB containing appropriate antibiotic with 0.5 mL overnight culture of E. coli containing the desired plasmid. Grow at 37℃ for 12-16 hours with vigorous shaking. (Do not use over 80 mL culture).

2. Harvest the bacterial by centrifugation at 5,000 x g for 10 minutes at room temperature. Pour off the supernatant and blot the inverted tube on paper towels to remove residual medium.

3. Add 4 mL Buffer A1 (Add RNase A into Buffer A1 before use) and completely resuspend bacterial pellet by vortexing or pipetting (Complete resuspension is critical for optimal yields).

4. Add 4 mL Buffer B1, mix gently but thoroughly by inverting 5 times and incubate for 2-5 minutes to obtain a slightly clear lysate. Do not incubate longer than 5 minutes. Over-incubating causes genomic DNA contamination and plasmid damage.

5. Add 5 mL Buffer C1, mix immediately by inverting 5 times and vortex for 10 seconds. NOTE: It is critical to mix the solution well, if the mixture still appears conglobated, brownish or viscous; more mix is required to completely neutralize the solution.

6. Transfer the lysate to a high speed centrifuge tube and centrifuge at 13,000 rpm (14,000-18,000 x g) for 20 minutes at room temperature! NOTE: If the rotor is cold, incubate the lysate at room temperature for 10 minutes and then perform centrifugation as described.