DNA isolation Flashcards
What type of specimens are used to extract DNA?
Blood Buffy coat Bone marrow Solid tissue Lavage fluids Bacteria, viruses Fungi Organelles, mitochondria
How is bone marrow and peripheral blood prepared for dna extraction?
Through density gradient centrifugation, differential osmolysis and use of streptokinase
How is tissue prepared for dna extraction?
It is freezed or crushed, minced, enzymatic digestion
How are plants/fungi prepared for dna extraction?
They’re homogenized, and vortex used with glass beads
What anticoagulants are used to collect blood sample for dna extraction?
EDTA (purple top) or acid citrate dextrose (yellow top)
Why is heparin not used?
It can inhibit PCR
What type of sample is preferable for microbe or viral nucleic acid?
Liquid serum or plasma with no cells
List the four dna extraction methods?
Organic, inorganic, solid phase, and crude methods
What is organic extraction method?
It’s extraction methods that uses organic chemicals, phenol, chloroform.
What is inorganic dna extraction methods?
It’s extraction method that uses inorganic chemicals, detergents, EDTA, acetic acid, salt (salting out, spooling)
What is solid phase dna extraction method?
It’s the extraction method in which DNA is immobilized on a solid support, beads, or columns.
What is crude extraction method?
This is the extraction method in which DNA is released from cell lysis, but not purified
What is the first step in organic extraction? What does this step do?
Cell lysis. It releases DNA from cells.
What does cell lysis require?
Enzymes and detergents
What do enzymes do in cell lysis?
Enzymes break down proteins or cell walls of bacteria
What proteins do enzymes break during organic dna extraction?
Protease
Proteinase K
Lysozyme
What is the role of detergent in cell lysis? Give examples of these detergents?
Detergents disturb cell membranes. SDS, TX-100
What is the second step in organic extraction after cell lysis?
Addition of the organic phase
What is the organic phase added in organic extraction?
PCI : Phenol, chloroform, isoamyl alcohol
In what proportion is PCI added in organic extraction?
25:24:1
What is the role of phenol, chloroform and isoamyl alcohol in organic extraction?
Phenol is acidic
Chloroform helps define organic and aqueous phases
Isoamyl alcohol prevents foaming
What is done after the addition of the organic phase?
It is mixed and centrifuged, aqueous layer is extracted and DNA is soluble at that time
In what layer is DNA soluble?
Aqueous layer
What does solid phase dna isolation consist of?
Binding dna to a solid column matrix or bead under high salt conditions
What are the most common columns used?
Spin column
List the steps in the solid phase dna isolation
Cell lysis
Adding cells to column, spin
Wash, spin
Add elution buffer and spin
In organic extraction, what is done after transferring aqueous phase to a new tube?
Adding alcohol and high salt concentration
What is the use of alcohol and the Hugh salt concentration in organic extraction?
DNA is insoluble in alcohol and the high salt promotes precipitation for dna to turn solid in solution
What happens when dna is added to column in solid phase dna isolation?
DNA binds and waste flows through
What is the wash and spin step for?
Removing contaminants from column
What happens after elution buffer is added?
DNA is realeased from column into clean tube
What are the steps for crude dna extraction?
Mixing beads with specimen
Lysing cells by boiling
Spin down debris and beads
DNA is in supernatant
What are the special considerations for RNA isolation?
RNA is dynamic so gene expression can change
RNA is temperature sensitive
RNA is very susceptible to degradation
How is RNA preserved?
RNA must be kept on ice when in use or frozen -80dC for storage
What are RNases?
These are enzymes that degrade RNA
How is DNA quality and quantity most determined?
Using spectrophotometry or fluorometry
What is the formula for DNA concentration?
A(260nm) * 50 ug/ml * dilution factor
How is RNA concentration calculated?
A(260nm) * 40 ug/ml * dilution factor
What is the maximum absorbance of nucleic acids?
260 nm
How is the quality for DNA or RNA calculated?
A260/A280
What is the optimum quality of DNA?
1.8 but 1.6 to 2.1 is acceptable
What is the optimum quality of RNA?
2.0 but 1.7 to 2.3 is acceptable
What is the maximum absorbance of proteins?
280nm
