Isolation of DNA
Transcript of Isolation of DNA
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Isolation of DNAIsolation of DNA
Group # 5Group # 5SIM, Michelle D.SIM, Michelle D.
SUDERIO, Gellina Ann R.SUDERIO, Gellina Ann R.TEOPE, Jonnah Kristina c.TEOPE, Jonnah Kristina c.TIMBOL, Danica Kaye P.TIMBOL, Danica Kaye P.UY, Regina Celine DG.UY, Regina Celine DG.
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DNA Isolation
• A routine procedure to collect DNA for analysis • 3 basic and one optional steps in a DNA
extraction – Cell disruption or cell lysis – Removing membrane lipids by adding a detergent – Adding a protease (optional but almost always
done) – Precipitating the DNA with an alcohol — usually ice-
cold ethanol or isopropanol
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• DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.
• DNA absorbs UV light at 260 and 280 nm
• Proteins absorb UV light at 280 nm – Pure DNA = 1.8– DNA with protein < 1.8
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Materials
1.5mL microcentrifuge tubes Water bath 800C Isopropanol (room temperature) 70% ethanol (room temperature) Nuclei lysis solution RNAse solution Protein Precipitation Solution Absorbent paper
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Isolation of Animal TissueAdd 3µL of RNase
Solution to the nuclear lysate
Add 200µL of Protein Precipitation Sol’n.
Centrifuge at 13,000-16,000 x g (4 min)
Formation of white pellet
•Invert the tube 2-5 times to mix sample
•Incubate mixture at 37۫C (15-30 min)
•Cool to room temp. (5 min)
•Vortex at high speed(20 seconds)
•Chill sample on(5 min)
•Remove supernatant
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Transfer DNA in 1.5mL microcentrifuge tube w/ 600µL
of room temp. isopropanol
White thread- like strands of DNA form a visible mass
Small white pellet visible (DNA)
•Invert tube to mix solution.
Centrifuge at 13,000-16,000 x g (room
temperature,1 min)
•Decant supernatant.
Isolation of Animal Tissue
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Isolation of Animal TissueAdd 100µL of room temp.
70% ethanol
•Invert tube several times (wash DNA)
Aspirate ethanol
Centrifuge at 13,000-16,000 x g (room
temperature,1 min)
•Invert tube on clean absorbent paper
Air- dry pellet (10-15 min)
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Add 100µL of DNA Rehydration Solution
Store DNA
(2-8 ۫C)
•Incubate at 65۫C for 1 hour (Rehydration of DNA)
Isolation of Animal Tissue
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Isolation of DNA from E. coli
Add 1mL overnight culture of Escherichia coli to a 1.5mL microcentrifuge tube
Centrifuge at 13,000 – 16,000xg for 2 minutes
Remove the supernatant
Add 600µL of Nuclei Lysis Solution gently until the cells are resuspended
Incubate at 800C for 5 minutes to lyse the cells
Cool to room temperature
Add 3µL of RNase Solution to the cell lysate
Invert the tube 2 to 5 times to mix the contents
Incubate at 370C for 15 – 60 minutes
Cooled to room temperature
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Add 200µL of Protein Precipitation Solution to the RNase - treated cell lysate
Vortex at high speed for 20 seconds
Incubate the sample for 5 minutes
Centrifuge at 13,000 – 16,000xg for 3 minutes
Transfer the supernatant containing the DNA to a clean 1.5mL microcentrifuge tube containing 600µL of room temperature
isopropanol
Gently mix by inversion until the white threadlike strands of DNA formed a visible mass
Centrifuge again for 2 minutes at 13,000 – 16,000xg
Carefully pour off the supernatant in a clean absorbent paper
Add 600µL of room temperature 70% ethanol
Gently invert the tube several times to wash the DNA
Centrifuge again for 2 minutes at 13,000 – 16,000xg
Isolation of DNA from E. coli
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Carefully aspirate the ethanol using a pipette
Place the pellet on a clean absorbent paper
Air dry for 10 – 15 minutes
Add 100µL of DNA rehydration solution to the tube to rehydrate the DNA by incubating at 650C for 1 hour or by incubating the solution overnight at 40Cor even at room
teperature
Isolation of DNA from E. coli
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Post Laboratory Questions1.) In the isolation of genomic DNA from human gall bladder with cancer. An
extraction buffer consisiting of 50mM Tris, pH 8, 25mM EDTA, 200 mM NaCl, 1% SDS was used. Why are these components included?
Tris buffer- for pH maintenance
EDTA- binds divalent metal ions (Ca2+, Mg2+, Mn2+) that could form salt with anionic PO43- group of DNA- destabilizes the cell membrane- prevents precipitation of DNA- inhibits DNAses
NaCl- loosens the cell wall for increase solubility and stability of DNA- releases the plasmid DNA and sheared cellular DNA- denatures the DNA of the cell
SDS- anionic detergent- disrupts ionic interaction between proteins
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Post Laboratory Questions
2.) Other reagents were also used during the isolation procedure. Give the role of:
• Chloroform– further denatures and coagulates the protein so
that they collect at interface between the aqueous the organic phase upon centrifugation
• 100% Ethanol– to precipitate, resuspend or recover the DNA
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Post Laboratory Questions
3.) What is the purpose of washing with 70% ethanol?
• To wash the pellets
4.) The DNA pellet is resuspended in TE buffer. Why? Can water be used instead of TE buffer? Why or Why not?
• No, because water will not allow the resuspension of the plasmid DNA