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]
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]
Protocol guide for the N. crassa yeast artificial chromosome library. Includes: Chromosome Walking; Hybridization screening of the YAC library; YAC restriction mapping and contig building; Preparation of chromosomal DNA plugs of YAC clones; Partial restriction enzyme digestion of YAC DNA plugs; Using CHEF gel analysis to resolve YAC clones; Southern Hybridization; Isolation of terminal restriction fragments from cloned DNA inserts in YAC clones; etc. - [Read Protocol Guide for the N. crassa Yeast Artificial Chromosome Library]
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]
Protocol describes a method for vasectomy in which the vas deferens is accessed through the abdominal wall. Mice are ready for mating after ~10-14 days. Vasectomized males can be bred with fertile females to obtain plugs for timed matings. The pseudopregnant females can then be used for oviduct and uterine transfers. For an alternative protocol, see Vasectomy for Generation of Sterile Males: Access via Scrotal Sac. - [Read Vasectomy for Generation of Sterile Males: Access via Abdominal Wall Protocol]
Protocol describes a method for vasectomy in which the vas deferens is accessed through the scrotal sac. Mice are ready for mating after ~10-14 days. Vasectomized males can be bred with fertile females to obtain plugs for timed matings. The pseudopregnant females can then be used for oviduct and uterine transfers. - [Read Vasectomy for Generation of Sterile Mouse Males: Access via Scrotal Sac Protocols]