Chapter 9-1. Nucleic Acid (DNA, RNA) Extraction and Purification Experiment
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1. Human DNA Extraction Experiment
2. Animal Cell DNA Extraction Experiment
3. Plant Cell DNA Extraction Experiment
4. Plasmid DNA Isolation from Bacteria Experiment
1. Human DNA Extraction Experiment
⑴ Prepare the sample for DNA extraction.
① Usually, blood, hair, or skin cells from the inside of the mouth are used.
⑵ Break the cell membrane and nuclear membrane to release DNA from the nucleus.
⑶ Use enzymes to prevent DNA degradation.
⑷ Prepare 15 μl of protease, place it in a microtube, and add 500 μl of the sample.
⑸ Incubate at 56°C to allow protein degradation, then add 500 μl of protein removal solution (P.C.I) and mix well.
⑹ Centrifuge at 13,000 rpm for 3 minutes and transfer only the supernatant to a new tube.
⑺ Add 1 ml of 100% ethanol to the tube and place it on ice to precipitate DNA.
⑻ Centrifuge at 13,000 rpm for 5 minutes and carefully discard only the supernatant.
⑼ Add distilled water to dissolve the DNA and transfer the DNA solution to an agarose gel.
⑽ Load the DNA mixture into the agarose gel and apply an electric field from the negative electrode (cathode) to the positive electrode (anode).
⑾ After a certain period, check the electrophoresis results, which are separated based on molecular weight, using a UV detection or fluorescence detection.
2. Animal Cell DNA Extraction Experiment
⑴ Wash cells with PBS solution.
① The process of removing animal cell culture media or other buffer solutions.
⑵ Suspend cells in STE solution.
① A buffer solution used in future experimental procedures.
⑶ Add 20% SDS solution.
① Dissolves the cell membrane.
⑷ Add proteinase K and incubate for 4 hours.
① Breaks down intracellular proteins.
⑸ Centrifuge.
① The supernatant is used in the next step.
⑹ Add phenol and mix.
① A process to dissolve proteins within cells.
⑺ Centrifuge.
① After centrifugation, three layers are visible: phenol layer at the bottom, protein layer in the middle, and the supernatant at the top.
② DNA is in the supernatant.
⑻ Add cold 95% ethanol and sodium acetate, then mix.
① This creates conditions for DNA precipitation.
② Adding these substances to a DNA-containing solution causes DNA to clump like a thread.
⑼ Centrifuge.
① DNA precipitates.
⑽ Add and mix 70% ethanol with the precipitate.
① A step to wash the DNA.
⑾ Centrifuge.
⑿ Dissolve the precipitate in an appropriate volume of buffer solution.
3. Plant Cell DNA Extraction Experiment
4. Plasmid DNA Isolation from Bacteria Experiment
⑴ Harvest bacteria.
⑵ Suspend in a solution containing 50 mM glucose, 10 mM EDTA, and 25 mM Tris-HCl (pH 8.0).
⑶ Add 0.2 N NaOH and 1.0% SDS solution, then mix.
⑷ Add 3 M potassium acetate solution (pH 4.8), then mix.
⑸ Centrifuge.
⑹ Add isopropanol, then mix.
⑺ Centrifuge.
⑻ Wash the precipitate with 70% ethanol.
⑼ Dissolve the precipitate in an appropriate volume of buffer solution.
5. RNA Extraction Experiment
⑴ Background Knowledge
① RNA is more susceptible to hydrolysis compared to DNA.
② There is an enzyme called RNAase in the body that destroys RNA.
⑵ 1st. Protecting RNA from RNA degradation.
① Use DEPC (diethylpyrocarbonate), an RNase (ribonuclease) inhibitor.
② DEPC is also used as a chemical modification reagent for proteins, ethoxyformylating amino groups and imidazole groups in proteins.
⑶ 2nd. Removing proteins.
① Method 1. Treat with phenol/chloroform: Denatures proteins and RNAase.
② Method 2. Use ultracentrifugation, etc.
⑷ 3rd. Selectively isolating RNA.
① Method 1. Salting-out
② Method 2. Multiple rounds of centrifugation or ultracentrifugation.
Input: 2019.03.06 20:13
Edited: 2020.02.08 12:54