By Peter B. Becker
Choice of protocols is designed to research the connection among chromatin constitution and serve as, and to clarify molecular mechanisms that keep watch over very important mobile capabilities comparable to transcription, replication, recombination, and DNA fix. tools hide issues from cell-free structures for the structure of chromatin heterogeneity in vitro, to options for in vivo research of protein-DNA interactions. equipment contain directions to make sure profitable replication and to prevent pitfalls.
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Additional info for Chromatin Protocols
4. The DNA fragments are purified by standard differential PEG/salt precipitation and DEAE ion-exchange chromatography. 5. DNA strands can be discriminated in the mapping method using radioactive phosphate isotopes by two methods: a. 5' endlabeling of both strands followed by digestion of DNA after mapping, using a restriction enzyme with its site close to one DNA terminus to remove the proximal label. b. 5' endlabeling of isolated strands separately and then reannealling before nucleosome assembly (see Note 4).
Single 5' Radioactive Modification of Purified DNA 1. 5 µg of a purified DNA fragment with the appropriate restriction endonuclease in the manufacturer’s buffer. 2. 5 vol of cold ethanol. 3. Resuspend the DNA in phosphatase buffer and treat with alkaline phosphatase for 1 h at 37°C. 4. 1% SDS, phenol extract the solution, and then precipitate the aqueous phase twice with ethanol and sodium acetate. 5. 5 µL of 10X T4 polynucleotide kinase buffer. 6. Add 50 µCi of [γ-32P]dATP and adjust volume to 24 µL with water.
In the 3' direction) (see Subheading 1. and Notes 14–15). 2. , circled “r” in Fig. 2A). 3. , base-pair –11 in Fig. 2A) (see Note 15). 4. Notes 1. Great care should be taken that nucleosome assembly and all subsequent mapping steps are done strictly at 0–4°C. We have observed a shifting of nucleosome positions upon heating for almost all nucleosomes we have studied, despite the fact that these were “positioned nucleosomes” (A. Maeder, K. Luger, A. Flaus, T. Rechsteiner, and T. J. Richmond, unpublished, [9,30,31]).
Chromatin Protocols by Peter B. Becker