Download - Isolation of DNA
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