dna isolation and gel electrophoresis
TRANSCRIPT
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DNA ISOLATIONAND
GEL ELECTROPHORESIS
Lucia Dhiantika Witasari
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Physical Characteristics of DNA
• DNA absorbs UV light at 260 nm– Allows quantitation
• DNA is water soluble• DNA precipitates in alcohols• DNA carries a net negative charge• DNA has characteristic melting & annealing temperatures
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DNA extraction – the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure•SDS, sarcosyl•CTAB (Cetyltrimethylammonium bromide)
•Proteinase K•Lysozyme
•Freezing•Sonication•Grinding
Chemical
Enzymatic
Mechanic
Alcohol •Ethanol•Iso-propanol
•Phenol•chloroform
•Sodium chloride•Sodium acetate
•Membrane•Beads
Organic solvents
Salt
DNA binding
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PLASMID DNA ISOLATION
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Lisis I, Lisis IILisis III
Fenol‐chloroform‐isoamilalkohol extraction
Et‐OH
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Gambaran saat penambahan SDS NaOH
Supernatan dipindahkan ke Eppendorf baru
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Sentrifugasi
DNA & RNADilarutkan TE
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CHROMOSOMAL DNA ISOLATION
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Extract and purify DNA
Resuspend & lyse bacterial cell walls.
– Resuspend cells in Ice cold TES(Tris, EDTA, NaCl)
– lysozyme (degrades peptidoglycan cell walls)
– Proteinase K (to degrade protein that are bound to the DNA molecule)
– Enzymes (RNAses ) are often added to degrade the RNA in the sample for purification
– SDS (detergent to lyse the cells)
Structure of a Gram‐Negative Cell Wall
The DNA in the nucleus is surrounded by proteins
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Detergent:
• Breaks apart membranes by attaching to the lipids (fats) & proteins in the membranes
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Extract and purify DNA
c. Separation of DNA from other molecules– Phenol extraction
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The cell and nuclear membranes have been broken apart,
as well as all of the organelle membranes,such as those around the mitochondria and
chloroplasts.So what is left?
• Proteins • Carbohydrates (sugars) • DNA
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DNA isolation
• DNA and RNA have similar physical properties
• Enzymes (RNAses) are often added to degrade the RNA in the sample for purification
• Enzymes (proteases) are also added to degrade protein that are bound to the DNA molecule
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Fluorescent Dye for DNA
• Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.
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GEL ELECTROPHORESIS• Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA.
• Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.
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• DNA is negatively charged.
+‐
Power
DNA
• When placed in an electrical field, DNA will migrate toward the positive pole (anode).
H O2
• An agarose gel is used to slow the movement of DNA and separate by size.
Scanning Electron Micrograph of
Agarose Gel (1×1 µm)
• Polymerized agarose is porous,
allowing for the movement of DNA
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+‐
Power
DNA
How fast will the DNA migrate?strength of the electrical field, buffer, density of agarose gel…Size of the DNA!*Small DNA move faster than large DNA…gel electrophoresis separates DNA according to size
smalllarge
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
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Gel types
• Agarose is isolated from seaweed and highly purified
• Mainly used for DNA size analysis and for DNA separation prior to bloting
• Percentage of agarose in gel affect size separation
• Increase % increased separation of small fragments
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Detection of DNA
• DNA can be detected by staining or labeling
• Commonly stained by adding ethidium bromide or silver staining
• Can be labeled with radioactive or fluorescent tagged nucleotides
• Often involves a blotting procedure
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100200300
1,650
1,000
500
850650
400
12,000 bp
5,000
2,000
DNA Ladder Standard
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.
-
+
DNAmigration
bromophenol blue
Note: bromophenolblue migrates at approximately the same rate as a 300 bpDNA molecule
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Kualitas DNAKonsentrasi tinggi
Utuh, tidak terputus‐putus
Tidak banyak terkontaminasi oleh protein
Menentukan Kualitas DNA
Dengan Spektrofotometer
Dilihat dengan elektroforesis, band yang tebal secara kualitatif menunjukkan DNA yang bagus
Analyzing DNA
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Wavelength of DNA
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Using SpectrofotometerConcentration of DNA
• 1 A260 Unit of dsDNA = 50 μg/ml
• 1 A260 Unit of ssDNA = 33 μg/ml
• OD value should range between 0.1 and 1.0 to ensure an optimal measurement.
Example of calculation:
• Volume of dsDNA sample: 100 μl• Dilution: 25 μl of sample + 475 μl H20 (1+19 dilution) • A260 : 0.44
• Concentration of dsDNA: 0.44 x 50 μg/ml x 20 = 440 μg/ml
• Amount of dsDNA: 440 μg/ml x 0.1 ml (=sample volume) = 44 μg 27dhiantika.staff.ugm.ac.id
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Purity of DNA
• Pure DNA: A260/A280 > 1.8
• An A260/A280 < 1.8 indicates that the preparation is contaminated with protein and aromatic substances.
• An A260/A280 > 2 indicates a ossible contamination with RNA.
• The OD gives no information about the size of the DNA
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