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Quantitation of DNA for Automated Sequencing Using the VersaFluor Fluorometer

Wenda Ribeiro and Margaret Saha, Department of Biology, College of William and Mary, Williamsburg, Virginia 23187


Introduction
DNA sequence analysis is an integral part of genetic research. Recently, the use of automated sequencers has become increasingly more common in small research laboratory settings. However, the success of automated long read sequencing reactions require precise picomolar concentrations of DNA. We have successfully employed the VersaFluor fluorometer (Bio-Rad) to accurately and routinely quantify DNA for sequencing with the LiCor 4200 automated sequencer.


Materials and Methods
A genomic clone for xHif (Xenopus hypoxia inducible factor) was isolated by a homology screen of a Xenopus muscle genomic library using a 32P-dCTP labeled mouse probe (a gift of Steven McKnight). Phage DNA was isolated using a standard protocol, digested with EcoRI and HindIII and run on a 1.2% agarose gel (Figure 1). Bands corresponding to insert DNA were excised and the DNA isolated on glass milk. The DNA fragments were subcloned into BlueScript SK+ (Stratagene) and transformed into DH5a cells. Plasmid DNA was isolated using anion exchange chromatography. A λZap (Stratagene) cDNA clone for Xenopus xHif was also isolated using a homology screen of a tadpole library. Rescued plasmid DNA was obtained using the manufacturers instructions and was subsequently transformed into DH5α cells. Plasmid DNA was isolated using anion exchange chromatography.

Quantitation of the DNA was achieved using the Fluorescent DNA Quantitation Kit (Bio-Rad) with a 360 nm excitation filter and a 460 nm emission filter. The
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Agarose, low melting/gelling temperature, high MW separation (>1000 bp), 25 g. Suitable for enzymatic modifications. Nuclease-free.Gel point: 24-30 C, EEO (-mr): 300 gm/cm2. Category: Nucleotides &...
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