PCR preparation of DNA inserts from lambda and plasmid vectors for RFLP mapping |
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Authors: | Charles J Simon Kirk M Schnorr |
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Institution: | (1) USDA/ARS Washington State University, 99164 Pullman, WA, USA;(2) Department of Crop and Soil Sciences, Washington State University, 99164 Pullman, WA, USA;(3) Laboratoire de Biologie Cellulaire, Institute National de la Recherche Agronomique, 78026 Versailles, France |
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Abstract: | A simplified method is described for preparing insert DNA for labelling reactions to be used in Southern hybridization. This
method works with sequences cloned into both plasmid and lambda phage, and eliminates many of the steps leading to the labelling
reaction. Small quantities of hostE. coli or lambda phage carrying a probe sequence are lysed and amplified via the polymerase chain reaction using standard sequencing
primers. Unincorporated nucleotides are removed by ethanol precipitation or gel purification and insert DNA is ready for radio-labelling.
This method reduces the time and expense associated with conventional insert preparation, and greatly simplifies the use of
sequences cloned into lambda phage. |
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Keywords: | RFLP mapping Southern hybridization PCR |
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