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 PO43group 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 to precipitate, resuspend or recover the DNA

100% Ethanol

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