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DNA Removal for Bioprocessing Applications

physiological salt solutions.


Experimental Design
The performance of TURBO DNase containing the new enhancer was compared to a competitors wild type DNase for removal of DNA from living cell cultures. The enzymatic removal of viral DNA in cultured cells was measured using real-time RT-PCR, and cell viability was assessed after DNase treatment. In these experiments, HeLa cells were grown to confluence for 72 hours (about 15,000 cells/well in a 96 well plate). Cell media were temporarily removed from the wells, pooled, and adjusted to the appropriate 1X buffer concentration with TURBO DNase Buffer (containing the new TURBO DNase enhancer) or the DNase Buffer supplied with the wild type DNase I. Then, either 5 units of TURBO DNase (11.6 ng), or 5 or 50 units of wild type DNase I was added (110 ng and 475 ng, respectively). The DNase-containing medium was then replaced onto the cells. After a 15 minute equilibration period at 37C, lambda genomic DNA was introduced at 200 ng/well. The DNase digestion reactions were incubated for 75 min at 37C, then the media were collected. DNase digestion products were concentrated 5-fold from the media by magnetic bead purification, and 5 l was used in real-time PCR with primer-probe sets for lambda genomic DNA (Figure 1). For viability studies, cells were treated with trypsin, resuspended in 175 l DMEM-H plus serum, and assayed by microcytometry on a Guava Technologies PCA-96 instrument.


Figure 1. TURBO DNase Eliminates More DNA Contamination than Wild Type DNase I. Even at a 10-fold lower unit concentration, TURBO DNase removed more DNA from living HeLa cell cultures. The table summarizes th
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