Fast and Large-Scale Plasmid DNA Prep Without Centrifugation

June 14, 2022

BlueskyReddit
MD Anderson Cancer Center - University of Texas
Cancer System Imaging (Diagnostic Imaging Division)
Lab Technician

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Company:

QIAGEN

Product Name:

HiSpeed Plasmid Kits

Catalog Number:

12662

One of our research is focused on B7-H3 (or CD276), a molecule of the B7 family, which may be a promising target for cancer treatment as is expressed on cancer cells. We were interested in scaling the production of a humanized antibody that is able to recognize B7H3. In particular that plasmids codifying for the Heavy and Light chain of the Antibody. For scaling the production of the antibody we needed to get related maxi-prep plasmids of good purity. The constructs were on the PCDNA 3.4 backbone and have Ampicillin resistance. We isolated pure plasmid DNA from 200 mL bacterial culture, in a concentration of ~400 ng/uL. Some of the advantages of this kit include: time-saving, no centrifuge needed so and so no risk of DNA loss in pellet.

Experimental Design and Results Summary

Application

For ultrafast purification of up to 750 µg transfection-grade plasmid

Starting Material

DH5α competent E. coli and plasmids with pcDNA 3.4 TOPO backbone(VH_AHF15948_hIg_GICH and VL_AHF15938_hIg_KCL , both with Amp R)

Tips

N/A

Results Summary

I processed the maxiprep and from 250mL of bacterial culture I obtained between 300 to 400 ng/uL of purified plasmids DNA.

DOI or PMID #

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Summary

The Good

Fast and large-scale plasmid DNA preparation without centrifugation

The Bad

N/A

The Bottom Line

HiSpeed Plasmid Kits provide fast, large-scale, anion-exchange-based plasmid DNA preparation without centrifugation. Up to 750 µg (maxi) high-copy plasmid DNA can be purified from 150 µl – 250 ml culture volumes. Tip design allows a very high flow rate, permitting DNA binding, washing, and elution steps for plasmid purification to proceed faster. LyseBlue buffer helps for optimum lysis and maximum DNA yield. Results: about 400 ng/uL of purified plasmid DNA.

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