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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.
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