1. Would histone digestion Mix work as Hypertonic buffer? 2. What is Precipitati

Question

1. Would histone digestion Mix work as Hypertonic buffer?

2. What is Precipitation Mix for?

3. From this lab, I prepared a "Zero digestion" sample (0 min. digestion) by excluding MNase. Is there another way I could have assembled this control sample?

Lab 1: Structure of DNA Lab 1: Structure of DNA Part l fve carbon Snr Introduction Deoxyribonucleic acid (DNA) is composed of repeating subunits called nucleotides. Each nucleotide consists of a five-carbon sugar, a phosphate group, and a nitrogenous base (either adenine, thymine, cytosine, or guanine). Structurally, DNA is an antiparallel double-helix with the individual strands held together by the pairing of complementary bases between the two strands (2, 3). The negatively charged phosphate groups of each nucleotide are on the outside of the helix giving the DNA molecule its atidic character (4, 5b In vivo, DNA is arranged in the context of chromatin - a term referring to the pnir contains two molecules each of H2A, H2B, H3 and H4. Approximately 150 base pairs , This structural unit of packaged nature of "free DNA" with histone octamers (Figure 1). The histone octamer (bp) of DNA is wrapped around each histone octame 1.7 times chromatin is called a nucleosome (two nucleosomes are shown above). If the H1 protein associates with a nucleosome it is now referred to as a chromatosome Chromatosomes compact the genome more than nucleosomes and are associated with 166 bp of DNA (Figure 2) (8, 9) Metaphase Figure 1. The Hierarchy of DNA Structure Double stranded DNA is arranged in varying levels of compacti vivo, ranging from the naked DNA Chromosome DNA fiber to the fully compacted 17.moi, metaphase chromosome. Nucleosomes provide the material on which DNA is wrapped, and the Cerical repetitive unit by which the genome // is ultimately arranged (8, 9). 30 nm Chromatin Fiber in o 198 t cancer 11 nm Nucleosome The starting material for tar lakes nA this lab is 0,1% NP-40 treated nuclei fróm HeLa eells HeLa et Co cels were incubated in a hypotonic buffe) and the nuclei were collected and washed Incubation in a hypertonic buffer extracted the saluble proteins from the nuclei, this produced a nuclear extract that can be used ik in vitro transcription assays, as well as other experiments that utilize nuclear proteins. The remaining extracted nuclei now only cells were incubated in a ssavs, as well a contain those proteins tightly bound to DNA - namely, the components of chroma 111 · Hela in Hypfonic hand yCei set art?

Explanation / Answer

1)

A hypertonic buffer contains salt concentrations more than the neighboring system. In this case, our buffer has elevated salt content, more than the nuclei so yes it can work as a Hypertonic buffer.

2)

Precipitation mix has ethanol and a salt as the chief ingredients. Ethanol and salts help to precipitate our DNA. The salt (generally sodium acetate) neutralizes charges on the DNA. Ethanol has less dielectric constant than water, thereby enabling the sodium bond DNA to clump together, making it less hydrophilic and ultimately precipitate out of the solution.

3)

MNase, being an enzyme is controlled by conditions that are required for proper functioning of an Enzyme - environment conditions of pH and temperature. If you do not add the buffer the enzyme will not work. If you keep the sample at elevated temperature the enzyme would denature (lose its functional 3D structure). In these ways, you can devise the control.

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