Pulsed Field Gel Electrophoresis Protocols Protocol Links
Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol Protocol for direct retrieval of DNA fragments from pulsed-field gels. A gel slice containing a fragment of DNA resolved by pulsed-field gel electrophoresis is treated with agarase. The released DNA can be used as a substrate for ligation or restriction without further purification.
Markers for Pulsed-field Gel Electrophoresis Protocol Protocol fopr markers of pulsed-field gel electrophoresis. Markers for pulsed-field gel electrophorsis can be generated by ligation of linear monomers of bacteriophage {lambda} DNA (48.5 kb) into a nested series of concatemers. This procedure yields a series of concatemers that contain up to 20 tandemly arranged copies of bacteriophage DNA.
Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of DNA from mammalian cells and tissues. Genomic DNAs from mammalian cells are prepared for pulsed-field gel electrophoresis by lysing cells in situ in an agarose plug. Following digestion with an appropriate restriction enzyme, the plug is loaded directly into the well of a pulsed-field gel or it can be melted before loading.
Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of intact DNA from yeast. Yeast cells are first treated enzymatically to break down the cell walls and then resuspended in low-melting-temperature agarose plugs. The DNA is liberated by infusing the plugs with lysis buffer and proteases. This method is used to prepare both conventional and artificial yeast chromosomes.
Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels Protocol Protocol for pulsed-field gel electrophoresis via contour-clamped homogeneous electric field gels. In CHEF gels, the electric field is generated from multiple electrodes, arranged in a square of hexagonal contour around the horizontal gel and clamped to predetermined potentials. Using a combination of low field strengths, low concentrations of aragose, long switching intervals, and extended periods of electrophoresis, DNAs up to 5000 kb can be resolved.
Restriction Endonuclease Digestion of DNA in Agarose Plugs Protocol Protocol for restriction endonuclease digestion of DNA in agarose plugs. Genomic DNA isolated from mammalian, yeast, or bacterial cells can be digested with restriction endonucleases by incubating agarose plugs containing the DNA in the presence of the desired enzyme. After digestion, the DNA can be fractionated by pulsed-field gel electrophoresis and either isolated from the gel or analyzed by Southern Hybridization.
Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration Protocol Protocol for retrieval of DNA fragments from pulsed-field gels following DNA concentration. DNA contained in a slice of low-melting-temperature agarose is first concentrated by electrophoresis into a high-percentage agarose gel, and then isolated by treatment with agarase. The resulting DNA preparation is purified by microdialysis.
