By Peter B. Becker
The cutting-edge tools accumulated listed here are designed to research the connection among chromatin constitution and serve as, and to explain the molecular mechanisms that regulate such very important mobile features as transcription, replication, recombination, and DNA fix. those confirmed equipment hide quite a lot of subject matters, from strong cell-free platforms for the reconstitution of chromatin heterogeneity in vitro, to either classical and state-of-the-art ideas for in vivo research of protein-DNA interactions. each one approach comprises particular step by step directions to make sure winning replication besides useful notes approximately the best way to stay away from pitfalls. All authors are best scientists, renowned for his or her methodological services within the chromatin study.
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Additional info for Chromatin Protocols (Methods in Molecular Biology Vol 119)
1. Mapping with Restriction Enzymes 1. 02 µg (DNA) of labeled cores in 20 µL of BT buffer with 5–10 U of restriction enzyme at 37°C for 20 min (see Note 15). 22 Studitsky Fig. 2. Mapping of positions of nucleosome cores before and after transcription by SP6 RNA polymerase with restriction enzymes. (Top) Restriction map of the 227-bp SacI-NcoI template. The template was labeled at the NcoI end. The cleavage sites for AseI, PacI, BglII, and HaeII, and a promoter for SP6 RNA polymerase are indicated.
Coli DNA Polymerase I 1. Incubate 10 µg DNA with 5 U of the Klenow fragment in 50 µL KLB buffer, supplemented with 100 µCi of α-32P-dNTP at 12°C for 5 min (see Note 2). 2. Add all four NTP to 100 µM and incubate at 20°C for 10 min. Stop the reaction by adding EDTA to final concentration 20 mM. 3. Extract the DNA with 1:1 (v/v) phenol:chloroform (see Note 3). 4. Precipitate the DNA with ethanol, wash with 70% (v/v) ethanol, dry, and dissolve in 25 µL HE. 5. Measure the dpm/µL by scintillation counting and its absorbance at 260 µm.
Freeze the filtration tubes containing the agarose plugs on dry ice for 15 min. 14. Spin down the agarose in a microfuge at maximum speed for 30 min at room temperature. The fluid from the agarose matrix will be collected in the 2 mL centrifuge tube surrounding the filtration device. 15. Gently remove the agarose plug from the bottom of the filtration device and place into a clean eppendorf tube. Save the centrifugation devices for use later. 16. 1% SDS and continue to crush the agarose. 17. After the agarose is crushed into tiny pieces, place all samples at 4°C overnight or for several hours.
Chromatin Protocols (Methods in Molecular Biology Vol 119) by Peter B. Becker