Good Quality Large-Scale Plasmid Isolation Kit

August 04, 2015

University of Tennessee HSC
Physiology
Postdoctoral Researcher

Overall

Quality of Results

Ease-of-Optimization

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Company:

QIAGEN

Product Name:

EndoFree Plasmid Maxi Kit (10)

Catalog Number:

12362

EndoFree Plasmid Maxi Kit from QIAGEN was used to isolate 7.5kb mammalian overexpression plasmid. The purpose of plasmid purification was to overexpress insert protein in mammalian cell lines in vitro and mouse in vivo. Isolated plasmid showed high (around 2.1) 260/280 and 260/230 ratio. Upon transfection of isolated plasmid in mammalian cell line low cytotoxicity was showed. This indicates plasmid was endotoxin free. Overall this Plasmid Maxi Kit showed the best result.

Experimental Design and Results Summary

Application

Mammalian cell transfection for protein overexpression

Starting Material

Transformed E.coli

Protocol Overview

Positive clones from transformed E.coli were allow to grow overnight in 250ml nutrient broth. Bacterial cells were pelleted by centrifugation and suspended in P1 buffer provided with the kit. Further steps were followed as per manufacturer's instruction.

Tips

Always ensure complete bacteria lysis

Results Summary

Final plasmid pellet were dissolved in 1ml of nuclease free water which gives a DNA concentration of 1.5 micrograms per microliter. 260/280 and 260/230 ratio was found to be >2.1. Endotoxin level was found to be very low in isolated plasmid.

Additional Notes

None

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Summary

The Good

Fast, gives good yield

The Bad

Prepacked Qiagen-tip column provided with kit, got blocked when I passed bacterial cell lysate.

The Bottom Line

Overall a good kit for plasmid isolation on a large scale. Downstream uses of plasmid was satisfactory. I recommend this product.

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