Why to neutralise chargs of dna during dna extracyoom?
Answers
Answer:
Yield and quality are fundamental features for any researchers during nucleic acid extraction. Here, we describe a simplified, semi-unified, effective, and toxic material free protocol for extracting DNA and RNA from different prokaryotic and eukaryotic sources exploiting the physical and chemical properties of nucleic acids. Furthermore, this protocol showed that DNA and RNA are under triple protection (i.e. EDTA, SDS and NaCl) during lysis step, and this environment is improper for RNase to have DNA liberated of RNA and even for DNase to degrade the DNA. Therefore, the complete removal of RNA under RNase influence is achieved when RNase is added after DNA extraction, which gives optimal quality with any protocols. Similarly, DNA contamination in an isolated RNA is degraded by DNase to obtain high-quality RNA. Our protocol is the protocol of choice in terms of simplicity, recovery time, environmental safety, amount, purity, PCR and RT-PCR applicability.
The positively ions of saturated salt solution neutralize the negatively charged phosphate groups of the DNA and RNA backbone. ... Yield and quality are the ultimate goal for any researchers during DNA extraction procedure. Doubtless, the quality increases by getting RNA free of DNA contamination Reagents
Proteinase K, 100% Ethanol, 70% Ethanol, Double distilled (DD) water, Ethylene diamine tetra acetic acid (EDTA), RNase, DNase, Pyrex beads, Agarose, Deoxyribonucleic acid (DNA) Marker, 2 × EasyPfu PCR SuperMix, 10% Sodium dodecyl sulfate (SDS), Glacial acetic acid (CH3COOH), Hydrochloric acid (HCl) and Sodium hydroxide (NaOH).
2.2. Equipments
Mortar, Pestle, PCR machine, Microscope, Refrigerated Benchtop centrifuge (MIKRO200R, Germany), Weighing scale, Pipettes (20, 100, and 1000 μl), 15 and 50 ml falcon tubes, 50 ml centrifuge tubes and Disposable Polypropylene micro-centrifuge tubes
2.3. Reagent setup
Tris buffer, Tris-EDTA (TE), DEPC-treated water, Saturated salt solution (NaCl), Neutral saturated salt solution, Acidic saturated salt solution and Lysis buffer:1X STE buffer (50 mM NaCl, 50 mM Tris-HCl and 100 mM EDTA; PH 8.0)
2.4. Procedure
2.4.1. Grinding in liquid nitrogen (Mortar and pestle)
2.4.1.1. DNA extraction protocol
Hepatic DNA extraction from mouse can be divided into six steps. These are:
2. Homogenization
1 g of the liver was taken and cut into pieces then ground using a porcelain mortar and pestle in 3 ml of lysis buffer containing 900 μl of 10% SDS. The emulsion was transferred to micro-centrifuge tubes and 100 μg proteinase K was added per ml of emulsion solution, and incubated for 1 h at 50 °C.
3 Phase separation
350 μl of neutral saturated salt solution (NaCl) per ml was added to the previous emulsion, the micro-centrifuge tube was capped and shaken gently by hand for 15 s, and then incubated at room temperature for 10 min. The micro-centrifuge tube was centrifuged at 590 × g for 15 min at room temperature with DNA remaining exclusively in the aqueous phase