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Nucleic Acid Extraction Sahil
Nucleic Acid Extraction Sahil
Nucleic Acid Extraction Sahil
SAHIL KULKARNI
SENIOR RESEARCH FELLOW
HAFFKINE INSTITUTE
Where is DNA present in the cell????
Purpose
To release nucleic acid from the cell
for use in other procedures
Must be free from contamination with
protein, carbohydrate, lipids or other
nucleic acids.
Used pure nucleic acids for testing.
Isolation
Routinely isolated from human, fungal, bacterial and
viral sources.
Pre treat to make nucleated cells available,
whole blood
Tissue samples
Microorganisms
Need sufficient sample for adequate yield.
There are three basic and one optional steps in a
DNA/RNA extraction:
•Breaking the cells open, commonly referred to as cell
disruption or cell lysis, to expose the DNA/RNA within. This is
commonly achieved by grinding or sonicating the sample.
Column purification
Phenol-chloroform extraction
•It is a liquid-liquid extraction technique
in biochemistry.
•Liquid-liquid extraction, also known
as solvent extraction and partitioning,
is a method to separate compounds
based on their relative solubilities in
two different immiscible liquids. It is
an extraction of a substance from one
liquid phase into another liquid phase.
•It is widely used in molecular biology for
isolating DNA, RNA and protein.
•Equal volumes of a phenol : chloroform
mixture and an aqueous sample are mixed,
forming a biphasic mixture.
How it works
•This method relies on phase separation by centrifugation of a mix of the
aqueous sample and a solution containing water-saturated phenol,
chloroform and a chaotropic denaturing solution (guanidinium
thiocyanate) resulting in an upper aqueous phase and a lower organic
phase (mainly chloroform).
• Nearly all of the RNA is present in the aqueous phase, while DNA and
protein partition in the interphase and organic phase, respectively.
•In a last step, RNA is recovered from the aqueous phase by precipitation
with 2-propanol or ethanol.
•DNA will be located in the interphase thus the technique can be used for
DNA purification alone.
•Guanidinium thiocyanate denatures proteins, including RNases, and
separates rRNA from ribosome and dna from histones.
Kit contains :
• Mini spin column
• Collection tube
• Buffers AVL
• AW1
• AW2
• AVE
• Carrier RNA
Procedure
•Pipette 560 μl of prepared Buffer AVL containing
carrier RNA into a 1.5 ml micro centrifuge tube.
•Add 140 μl body fluid to the Buffer AVL carrier RNA
Mix by pulse vortexing for 15 s.
•Incubate at room temperature (15-25°C) for 10 min.
•Briefly centrifuge the tube to remove drops from the
inside of the lid.
Role of AVL
•The sample is first lysed under the highly denaturing
conditions provided by buffer AVL to inactivate RNases
and to ensure isolation of intact viral RNA
Why we add carrier RNA?
Firstly , it enhances binding of viral nucleic acids to the
QIAamp mini membrane, especially if there are very few
target molecules in the sample.
Secondly the addition of large amounts of carrier RNA
reduces the chance of viral RNA degradation in the rare
event that Rnase molecules escape denaturation by
chaotropic salts and detergents in buffer AVL.
If carrier RNA is not added to buffer AVL this may lead to
reduced viral RNA recovery.
•Add 560 μl of ethanol (96-100%) to the sample, and
mix by pulse-vortexing for 15 s.
only ethanol should be used since other alcohols may result in reduced
RNA yield and purity.
•After mixing, briefly centrifuge the tube to remove
drops from the lid.
•Carefully apply 630 μl of the solution to the Mini Spin
Column-in a 2ml Collection tube-without wetting the
rim.
•Close the cap, and centrifuge at 8000 rpm for 1 min.
•Place the spin column into a clean 2ml collection tube
and discard the tube containing the filtrate.
•Carefully open the Mini Spin column and repeat the
step .
•Carefully open the Mini Spin Column and add 500 μl of
Buffer AW1.
WASH BUFFER- PROTEIN DENATURING CHEMICALS
•Close the cap and centrifuge at 8000rpm for 1 min.
•Place the Mini Spin Column in a clean 2 ml collection
tube and discard the tube containing the filtrate.
•Carefully open the Mini Spin Column and add 500 μl of
Buffer AW2.Close the cap and centrifuge at 13,000 rpm
for 4 min.
INCREASES THE AFFINITY BINDING OF RNA, AND CREATES AN
ENVIRONMENT FOR RNA BINDING TO SILICA SURFACE
•Place the Mini Spin Column into a clean, labeled 1.5ml
micro centrifuge tube.
• Discard the old collection tube containing the filtrate.
• Carefully open the Spin Column & add 60 μl of Buffer AVE
equilibrated to room temperature.
It is elution buffer nothing but Rnase, Dnase free water.
• Close the cap and incubate at room temperature for 1 min.
Centrifuge at 8000rpm for 1 min.
•Stored the viral RNA at -20 to -70 degree.
•RNA loss is a major cause of extraction failure and commonly
occurs during extraction of RNA from samples using column.
•More than 30-40% of the RNA is lost due to insufficient binding
of the RNA, or incomplete elution of the RNA from the column.
•Hence, a non-binding, non-elution extraction method, which
eliminates the RNA loss and maximizes the yield of RNA should
be developed.
×2
Quantification:
•Abundance in weight: spectroscopic quantification
•Absolute abundance in number: Q-PCR
•Size: Gel electrophoresis
Synthesis:
•De novo: Oligonucleotide synthesis
•Amplification: PCR
Other Applications:
• Nucleic acid simulations
•DNA sequencing
•Expression cloning
•Southern blot
•northern blot
•Fluorescent in situ hybridization
•several Bioinformatics methods, such as RNA structure prediction
THANK YOU
sahilkulkarni@gmail.com