Midiprep Yields in a Miniprep Format

April 02, 2018

BlueskyReddit
Duke University
Immunology
Graduate student

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Omega Bio-Tek

Product Name:

E.Z.N.A. Endo Free Plasmid Mini Kit II

Catalog Number:

D6950

I needed ~100 ug of endotoxin-free plasmid DNA for retrovirus production. However, without a good vacuum manifold, midipreps are such a hassle. With the EZNA Endo Free Mini Kit II, one can easily get 100 ug of DNA from a 12 mL bacterial culture using a simple tabletop microcentrifuge, and in much less time than a typical midiprep.

Experimental Design and Results Summary

Application

Endotoxin-free plasmid miniprep or midiprep

Starting Material

NEB Stable cells harboring pMX-derived retroviral plasmids

Protocol Overview

Inoculate 12 mL LB broth with single colony from agar plate. Incubate overnight with vigorous shaking. Pellet bacteria by centrifugation. Follow manufacturer's instructions (resuspension, lysis, neutralization, clarification, silica column-based purification). There are more wash buffers and wash steps than a typical miniprep -- this is to eliminate endotoxin. Elute twice in 50 uL elution buffer; pool eluate for 100 uL of high-concentration (0.5-1.1 ug/uL) plasmid DNA.

Tips

Elute the columns twice (in 50 uL elution buffer each time) for high yield and high concentration.

Results Summary

50-100 ug of plasmid DNA per 12 mL culture.Routinely, A260/A280 = 1.85 (little/no protein contamination). A260/A230 is highly variable (0.7-2.0) within a single experiment, even for scientists with extensive experience in plasmid preps. The DNA works well for transfection experiments. It is possible that the high A230 measurement is due to contamination by silica fine particles from the column (This is suggested as a possibility in the troubleshooting section of the product manual).

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Very high yield of (purportedly) endotoxin-free DNA from a miniprep kit. Faster, easier, and much cheaper than midiprep kits.

The Bad

The highly variable A260/A230 reading lowers confidence in the results, but there are no drawbacks for downstream applications.

The Bottom Line

Our lab has switched from midipreps (more expensive and time-consuming) to this miniprep kit to isolate 50-100 ug yields of endotoxin-free transfection-ready DNA.

Join the discussion