Much Room for Optimization

June 08, 2016

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Quality of Results

Ease-of-Optimization

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Company:

Promega

Product Name:

Pure Yield Plasmid Midiprep Kit

Catalog Number:

A2495

The Pure Yield Plasmid Midiprep Kit relies on columns that fit either a common 50-ml conical tube or a vacuum manifold, and would therefore save you lots of time in comparison to other kits with DNA affinity columns that work by gravity and need a final centrifugation step in a floor centrifuge. The purification steps are, unfortunately, not efficient and you end up losing more time than you anticipated you would spare, and for poor overall yield compared to other commercial kits.

Experimental Design and Results Summary

Application

Plasmid DNA prep

Starting Material

E. Coli overnight liquid culture (20-100 ml)

Protocol Overview

Alkaline lysis is performed as is common with other kits, then lysate is cleared by passing through a filter column (clogs easily and you might need to repeat the procedure changing filter), then lysate is passed through the affinity column (a cloudiness in the lysate might need another step of centrifugation to make it eventually clear), then you wash with an endotoxin removal buffer and a wash buffer. Drying the column requires much effort as it remains slightly wet and you risk carryover of wash buffer in the final elution. Elution is performed by centrifugation or via vacuum (would require an adaptor that is bought separately) and the final DNA volume still needs clearing by some white suspension that remains in the eluate (see image).

Tips

You need to clear your lysate in a floor centrifuge not to have it clog the membrane prior to filtration step. Once you elute DNA, make sure you spin it for 5' at 13000 rpm in a minifuge, or it will be cloudy and possibly perform poorly in downstream applications

Results Summary

DNA yield is poor compared even to a miniprep column for a comparable amount of overnight culture. From 100 ml culture, I got an average of 150-200 ug plasmid DNA, which is a pale comparison to 50ug plasmid DNA from a 20ml culture processed with a simple miniprep kit from other vendors.

Additional Notes

The kit comes without the ethanol and isopropanol needed for the endotoxin removal and wash buffer, respectively. The unopened neutralization buffer bottle was corroded by the buffer (see image). If you use the vacuum manifold (bought separately), the adaptors are not perfectly airtight, especially after some rounds of use.

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Summary

The Good

The kit is designed to spend less time on column filtration and does not necessitate a floor centrifuge.

The Bad

Poor yield and sample handling is more complex than it would seem

The Bottom Line

This is a kit suitable to experienced users that need to process dozen of samples at a time and do not have much hands-on time to devote to their preps. They should also be willing to sacrifice yield and purity for speed.

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