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Isolation of DNA
Isolation of DNA
Group # 5
SIM, Michelle D.
SUDERIO, Gellina Ann R.
TEOPE, Jonnah Kristina c.
TIMBOL, Danica Kaye P.
UY, Regina Celine DG.
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
• 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
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
Isolation of Animal Tissue
Add 3µL of RNase
Solution to the nuclear
lysate
•Invert the tube 2-5 times to mix sample
•Incubate mixture at 37۫C (15-30 min)
•Cool to room temp. (5 min)
Centrifuge at
13,000-16,000 x g (4 min)
•Remove supernatant
Isolation of Animal Tissue
Transfer DNA in 1.5mL
microcentrifuge tube w/ 600µL
of room temp. isopropanol
Centrifuge at
13,000-16,000 x g (room
temperature,1 min)
•Decant supernatant.
Isolation of Animal Tissue
Add 100µL of room temp.
70% ethanol
Centrifuge at
13,000-16,000 x g (room
temperature,1 min)
Aspirate ethanol
Store DNA
(2-8۫C)
Isolation of DNA from E. coli
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
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
Post Laboratory Questions
3.) What is the purpose of washing with 70%
ethanol?
• To wash the pellets