pulsed Protocols

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol]

Category: Transfection Protocols: Stable Transfection Protocols

Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate-DNA coprecipitation. But, as with other transfection methods, the optimal conditions for electroporating DNA into untested cell lines must be determined experimentally. - [Read DNA Transfection by Electroporation]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to separate chromosomes and restriction fragments using pulsed field gel electrophoresis. - [Read How to Separate Chromosomes and Restriction Fragments Using Pulsed Field Gel Electrophoresis]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes the use of agarose plugs for isolation of yeast artificial chromosome (YAC) DNA. The DNA can then be run on a pulsed-field gel and used for microinjection to produce transgenic mice. - [Read Large-Scale Preparation of Agarose Plugs of Yeast DNA Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Markers for Pulsed-field Gel Electrophoresis Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for pulsed-field gel electrophoresis via transverse alternating field electrophoresis gels. - [Read Pulsed-field Gel Electrophoresis via Transverse Alternating Field Electrophoresis Gels Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Restriction Endonuclease Digestion of DNA in Agarose Plugs Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

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. - [Read Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration Protocol]