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)

Add 200µL of Protein

Precipitation Sol’n.
•Vortex at high speed(20 seconds)
•Chill sample on(5 min)

Centrifuge at
13,000-16,000 x g (4 min)

Formation of white pellet

•Remove supernatant
Isolation of Animal Tissue
Transfer DNA in 1.5mL
microcentrifuge tube w/ 600µL
of room temp. isopropanol

•Invert tube to mix solution.

White thread- like strands of

DNA form a visible mass

Centrifuge at
13,000-16,000 x g (room
temperature,1 min)

Small white pellet visible

(DNA)

•Decant supernatant.
Isolation of Animal Tissue
Add 100µL of room temp.
70% ethanol

•Invert tube several times (wash DNA)

Centrifuge at
13,000-16,000 x g (room
temperature,1 min)

Aspirate ethanol

•Invert tube on clean absorbent paper

Air- dry pellet (10-15 min)

Isolation of Animal Tissue
Add 100µL of DNA
Rehydration Solution

•Incubate at 65۫C for 1 hour (Rehydration of

DNA)

Store DNA
(2-8۫C)
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
Isolation of DNA from E. coli
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
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
 Post Laboratory Questions
1.) 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
 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

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