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.

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 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

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

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)

Add 100µL of DNA Rehydration Solution

Store DNA

(2-8 ۫C)

•Incubate at 65۫C for 1 hour (Rehydration of DNA)

Isolation of Animal Tissue

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

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

Isolation of DNA from E. coli

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

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