Lvris and Irelation of DNA from Ranana)

Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
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DNA Extraction by Alkaline Lysis
Procedure:
1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to
pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip.
The spins can be performed at 4C or at room temperature. Longer spins make it difficult
to resuspend cells.
2. Resuspend pellet in 100µl GTE solution and let sit 5 min at room temperature. Be sure
cells are completely resuspended.
3. Add 200µl NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5
min.
4. Add 150µl potassium acetate solution and vortex at maximum speed for 2s to mix.
Place on ice for 5-15 min. Be sure mixing is complete.
5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA.
6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4
ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids.
7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and RNA.
8. Remove supernatant, wash the pellet with 70% ethanol, and dry pellet under vacuum.
9. Resuspend the pellet in 30ul TE buffer and store at 4°C. Contaminating RNA may
interfere with detection of DNA fragments on the agarose gel; it can be destroyed by
adding lul of a 10 mg/ml RNase solution (DNase-free).
Isolation of DNA from Banana
Procedure:
1) The precipitating agent was prepared by adding 1 tablespoon of salt and 5 tablespoons
ofliquid soap. Then, 100mL of hot water was mixed and stirred evenly.
2) A banana was peeled and chopped into small pieces, then it was placed in a bowl
withsufficient water and then mixed with the precipitating agent.
3) The banana was then mashed with a fork.
4) The mashed banana was then filtered through gauze with a strainer and the
filtrate iscollected in a transparent container.
5) The methylated spirit was then taken out from the fridge and mixed with the filtrate
gently,and the sample is observed for changes for 2-3 minutes.
6) The methylated spirit was then taken out from the fridge and mixed with the filtrate
gently,and the sample is observed for changes for 2-3 minutes.
Compare and contrast the difference between this two protocol (DNA Extraction by Alkaline
Lysis and Isolation of DNA from Banana)
Transcribed Image Text:DNA Extraction by Alkaline Lysis Procedure: 1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip. The spins can be performed at 4C or at room temperature. Longer spins make it difficult to resuspend cells. 2. Resuspend pellet in 100µl GTE solution and let sit 5 min at room temperature. Be sure cells are completely resuspended. 3. Add 200µl NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5 min. 4. Add 150µl potassium acetate solution and vortex at maximum speed for 2s to mix. Place on ice for 5-15 min. Be sure mixing is complete. 5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA. 6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4 ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids. 7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and RNA. 8. Remove supernatant, wash the pellet with 70% ethanol, and dry pellet under vacuum. 9. Resuspend the pellet in 30ul TE buffer and store at 4°C. Contaminating RNA may interfere with detection of DNA fragments on the agarose gel; it can be destroyed by adding lul of a 10 mg/ml RNase solution (DNase-free). Isolation of DNA from Banana Procedure: 1) The precipitating agent was prepared by adding 1 tablespoon of salt and 5 tablespoons ofliquid soap. Then, 100mL of hot water was mixed and stirred evenly. 2) A banana was peeled and chopped into small pieces, then it was placed in a bowl withsufficient water and then mixed with the precipitating agent. 3) The banana was then mashed with a fork. 4) The mashed banana was then filtered through gauze with a strainer and the filtrate iscollected in a transparent container. 5) The methylated spirit was then taken out from the fridge and mixed with the filtrate gently,and the sample is observed for changes for 2-3 minutes. 6) The methylated spirit was then taken out from the fridge and mixed with the filtrate gently,and the sample is observed for changes for 2-3 minutes. Compare and contrast the difference between this two protocol (DNA Extraction by Alkaline Lysis and Isolation of DNA from Banana)
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