Why use sodium acetate in DNA extraction?
In DNA precipitation, a salt (sodium acetate) reacts with DNA. It breaks up into Na+ and (CH3COO)–. The positively charged sodium ion neutralize negatively charged PO3– of the DNA. Hydrophilic nature of DNA helps it to dissolve it in water but by reacting with sodium acetate, DNA becomes less hydrophilic.
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What is the effect of PEG on DNA fragments of different sizes?
By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained.
What does PEG nacl do?
Polyethylene glycol (PEG) has a wide range of uses including cell fusion for the formation of hybridomas, precipitation of DNA, and to create macromolecular crowding in solutions.
How does PEG precipitate DNA?
In it’s simplest form, a solution containing salt and a particular concentration of PEG (normally PEG8000) is added to an aqueous nucleic acid sample, which is then incubated. This results in precipitation of the nucleic acids, which are then pelleted by centrifugation and then washed twice in 70% ethanol.
How does salt affect DNA extraction?
Salt is used during the process of DNA extraction because salt helps in neutralizing DNA molecules, which makes the DNA molecule less hydrophilic, i.e. less soluble in water. Salt also helps in detaching protein molecules bound to the DNA. By which, the extraction process will be easier.
Which chemical is used to precipitate DNA during the procedure of DNA isolation?
Chilled ethanol
So, the correct answer is ‘Chilled ethanol’.
How does DNA size selection work?
DNA size selection (or DNA sizing) is the targeted capture of DNA fragments of a specific size or a size range. It’s broadly used in sequencing. And it’s a sample preparation step that has the greatest impact on the quality of sequencing results.
How does size selection with AMPure beads work?
Size selection First a low concentration of AMPure XP beads is added to the sample to bind larger DNA fragments. In this step the beads containing the larger fragments are discarded. More beads are then added to the supernatant, increasing the amount of PEG and NaCl, so smaller fragment sizes will be bound.
What is PEG precipitation?
Precipitation by polyethylene glycol (PEG) is a common method to concentrate and capture large proteins as the first step of a purification process. The phenomenon could be interpreted through the theories of attractive depletion (Asakura and Oosawa, 1958) and excluded volume (Iverius and Laurent, 1967, Polson, 1977).
How does PEG concentration work?
What is purpose of using salt in extraction process?
How does salt and alcohol help in DNA precipitation?
The overall function of salt and ethanol/ isopropanol is to precipitate DNA from the solution. The salts neutralize the negative charge of the negatively charged phosphate in DNA and the isopropanol /ethanol removes the hydration shell of H2O molecules around the phosphate.
Why is sodium acetate used in DNA precipitation by ethanol rather than sodium chloride?
Use sodium acetate (0.3 M final conc, pH 5.2) for routine DNA precipitation. Use sodium chloride (0.2 M final conc) for DNA samples containing SDS, since NaCl keeps SDS soluble in 70% ethanol so that it doesn’t precipitate with the DNA.
Why is DNA size important?
Precise DNA sizing can boost sequencing efficiency, save money, improve data, and even allow sequencing of low-input samples.
How does size selection with beads work?
What do AMPure beads do?
AMPure beads work like this: DNA has a negatively charged phosphate backbone and in a solution with a lot of salt and polyethylene glycol (PEG) the DNA gets crowded out of solution. It ends up on the beads, and because they are magnetic you can collect them using a magnetic held on one side of the tube.
How does PEG protein precipitation work?
The effective protein concentration can thus be increased by increasing the concentration of PEG, until the critical concentration is exceeded, and precipitation occurs.
How do you make PEG NaCl?
PEG/NaCl (5x) stock solution: PEG-8000 20%, NaCl 2.5 M. Dissolve 100 g PEG-8000 (20% w/v) and 75 g NaCl (2.5 M) in 400 ml ddH20 and bring to a final volume of 500 ml by stirring at RT. The solution can be autoclaved (optional) but mixing during the cooling period is required to prevent a phase separation. Store at RT.
How does salt concentration affect DNA melting temperature?
The melting temperature increases logarithmically with the salt concentration of the solution. The more GC base pairs in the chain enhance the stability of DNA chain at a fix salt concentration.