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New Protocols for Isolating High- Molecular-Weight Genomic DNA

ts. However, other organic extraction protocols begin with a blood volume of 20 ml7 so the lack of DNA recovery may have been due to an insufficient starting blood volume. The dialysis method yielded enough high-molecular-weight DNA from the same blood volume (5 to 7 ml) to package an average of over 3 million pfu of shuttle vector per sample. Among the remaining tissues tested, packaging efficiencies averaged 16% higher with the RecoverEase DNA samples, compared to those prepared using organic extraction. Although this increase is modest compared to previous comparisons in tissues such as liver,6 note that bone marrow and cultured cells are among the best substrates for the organic extraction method in terms of high rates of packaging success. DNA yields were similar using both techniques. DNA purity was also indistinguishable, with A260/280 ratios of 1.8 among all samples (data not shown).

Conclusions

The list of tissues amenable to Stratagenes RecoverEase DNA isolation kit now includes blood, bone marrow, and cultured cell lines. With the kit, reduce the need for hands-on time, eliminate toxic organic solutions, and remove ethanol precipitation and lengthy rehydration steps. Other potential applications for genomic DNA recovery include Southern blot analysis, pulsed-field gel electrophoresis, PCR amplification, genomic library preparation, and shuttle vector recovery in transgenic in vivo mutation assays.

Methods8

Blood: Recover rodent whole blood (5 to 7 ml) with the anticoagulant ACD (0.48% citric acid w/v, 1.32% sodium citrate w/v, 1.47% glucose w/v),7 and add five volumes of Tris-buffered ammonium chloride (140 mM NH4Cl, 17 mM Tris-HCl, pH 7.65). After 5 minutes at 37C, pellet the lysed blo
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RNase-Free DNase 50 l...
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