Extraction and purification of DNA:

DNA must be purified from cellular material in a manner that

prevents degradation. Because of this, even crude extraction
procedures can still be adopted to prepare a sufficient amount of
DNA to allow for multiple end uses.
DNA extraction from plant tissue can vary depending on the
material used. Essentially any mechanical means of breaking down
the cell wall and membranes to allow access to nuclear material,
without its degradation is required.
For this, usually an initial grinding stage with liquid nitrogen is
employed to break down cell wall material and allow access to
DNA while harmful cellular enzymes and chemicals remain
inactivated.
Once the tissue has been sufficiently ground, it can then be
resuspended in a suitable buffer, such as CTAB.
 In order to purify DNA, insoluble particulates are removed
through centrifugation while soluble proteins and other material
are separated through mixing with chloroform and centrifugation.
DNA must then be precipitated from the aqueous phase and
washed thoroughly to remove contaminating salts.
The purified DNA is then resuspended and stored in TE buffer or
sterile distilled water. This method has been shown to give intact
genomic DNA from plant tissue.
To check the quality of the extracted DNA, a sample is run on an
agarose gel, stained with ethidium bromide, and visualised under
UV light.
Procedure
 Grind 200 mg of plant tissue to a fine paste in approximately 500 μl of CTAB buffer.
 Transfer CTAB/plant extract mixture to a microfuge tube.
 Incubate the CTAB/plant extract mixture for about 15 min at 55 o C in a recirculating
water bath.
After incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to spin
down cell debris. Transfer the supernatant to clean microfuge tubes.
To each tube add 250 μl of Chloroform : Iso Amyl Alcohol (24:1) and mix the
solution by inversion. After mixing, spin the tubes at 13000 rpm for 1 min.
Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge
tube.
To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice cold
absolute ethanol.
Invert the tubes slowly several times to precipitate the DNA. Generally the DNA can
be seen to precipitate out of solution. Alternatively the tubes can be placed for 1 hr at -
20 o C after the addition of ethanol to precipitate the DNA.

Following precipitation, the DNA can be pipetted off by slowly rotating/spinning a

tip in the cold solution. The precipitated DNA sticks to the pipette and is visible as a
clear thick precipitate. To wash the DNA, transfer the precipitate into a microfuge tube
containing 500 μl of ice cold 70 % ethanol and slowly invert the tube. Repeat.
Do not allow the DNA to over dry or it will be hard to re-dissolve.
Resuspend the DNA in sterile DNase free water (approximately 50-400
μl H2O; the amount of water needed to dissolve the DNA can vary,
depending on how much is isolated).
RNaseA (10 μg/ml) can be added to the water prior to dissolving the
DNA to remove any RNA in the preparation (10 μl RNaseA in 10ml
H2O).
After resuspension, the DNA is incubated at 65o C for 20 min to
destroy any Dnases that may be present and store at 4o C.
Agarose gel electrophoresis of the DNA will show the integrity of the
DNA, while spectrophotometry will give an indication of the
concentration and cleanliness.
 DNA quality confirmation
Prepare a 1 % solution of agarose by melting 1 g of agarose in 100 mL of
0.5x TBE buffer in a microwave for approximately 2 min. Allow to cool for
a couple of
minutes then add 2.5 μl of ethidium bromide, stir to mix.
Cast a gel using a supplied tray and comb. Allow the gel to set for a
minimum of 20 min at room temperature on a flat surface.
Load the following into separate wells
* 10 μL 1kb ladder
* 5 μL sample + 5 μL water + 2 μL 6x Loading Buffer
Run the gel for 30 min at 100 V
Expose the gel to UV light and photograph (demonstration)
Confirm DNA quality, presence of a highly resolved high molecular
weight band
indicates good quality DNA, presence of a smeared band indicates DNA
degradation.
Principle of RNA extraction:
An RNA of a living cell (animal, plant, human, bacteria or virus)
provides information on transcriptional activity, thus, important for gene
expression studies. RNA lysis buffer lyses the cell wall/membrane while
the use of organic solvents like phenol-chloroform separates RNA upon
centrifugation.
Phenol: chloroform: isoamyl alcohol, by centrifugation, forms three
layers viz the lower organic layer, middle DNA and protein-containing
layer and upper aqueous RNA-containing layer. The upper aqueous
phase is collected, precipitated, washed and dissolved in RNase-free
water.
 The problem with RNA extraction:
 As aforementioned, there is a problem handling the RNA and restricts the
reproducibility of any RNA extraction method. Some common problems are
explained here,
 RNA is single-stranded and less stable than DNA.
 Heat and hydrolysis can degrade the sugar backbone of RNA easily.
 Less cellular quantity results in low yield.
 Less stability of RNA: The tissue should be immediately processed for extraction as
the time increases the chances that RNase will degrade RNA increases. Using
commercially available RNA stabilizers can help to extend RNA pre-processing time,
though.
 And most importantly, the universal presence of the RNase enzyme degrades RNA
more easily than DNA. RNase is predominantly present in the atmosphere,
surrounding environment, on a working desk or even on our hands degrades the
extracted RNA more easily.
 These reasons make the RNA isolation technique more sensitive and specialized
which needs extraordinary preparation and care, every time. The effective RNA
extraction protocol relies on these 5 things,
 Inactivation of RNase
 Stabilizing RNA
 Isolating RNA from DNA and protein.
 Precipitating only RNA
 Steps in RNA extraction :
 Sample collection
 Sample preparation
 Tissue homogenization
 Cell lysis
 RNA precipitation
 RNA washing
 RNA elution
 RNA quality check
 RNA quantification
 RNA integrity check
 Storing and handling
 Process of RNA extraction:
 Sample collection:
 Cells, cell lines, cultures and tissues are common sample types for RNA
extraction. The sample is isolated aseptically and stored at -80 ℃. It is
advisable to collect samples by an expert. In order to make RNA stable, the
tissues are stored at -80℃ before processing.
 Note that additives are added to store the tissue sample for a longer period
of time, as RNA starts losing its integrity under adverse environments.
 Sample preparation:
 Preparing a sample before RNA extraction is so crucial for higher yield and
greater purity. Oftentimes, the tissue homogenization technique is utilized
to prepare a homogenous mixture of cells.
 Usually, for plant tissue or solid mammalian tissues, a mortar and pestle
can be used (although professional tissue homogenizers are advisable).
During tissue grinding, RNA lysis buffer is carefully added, slowly to the
sample.
 Upon getting fine homogenization, the sample is ready for extraction.
Noteworthy, perform steps at 4℃ temperature and under the strict aseptic
hood. Take necessary precautions to avoid RNase activities .
 Sample quantity:
 Sample type : Concentration
 Tissue sample 50 to 100 mG
 Monolayer cell culture 1 × 105 –1 × 107
 Cell culture in suspension 5–10 × 106 cells from animal,
plant, or yeast origin or 1 × 107 cells of bacterial origin
 Optimization
 Quickly perform RNA isolation after sample collection,
RNA degrades rapidly, or store tissue sample immediately
into liquid nitrogen at -80℃ for long-term use.
 Cell lysis and chemical treatment:
 Chemical processing followed by centrifugation separates RNA from other
cell debris and chemical traces. Common chemicals are combinations of
various organic solvents that separate RNA into the upper aqueous phase.
 An additional step of DNase treatment can be applied to remove the DNA
from the sample, which eventually yields a good quality and highly pure
RNA.
 Precipitation:
 The RNA precipitation process is much similar to the
DNA precipitation process. Here using any alcohol the RNA is precipitated
into a visible form. However, to precipitate only RNA, a specialized agent
Lithium chloride is used.
 RNA washing:
 Precipitated RNA pellets are washed before dissolving and downstream
processing. Washing removes other contaminants and debris like DNA,
protein and traces of phenol and other chemicals. Again the process of
nucleic acid washing remains the same as DNA washing. Washing gives a
pure, whitish RNA precipitate.
 Dissolving RNA:
 RNA is dissolved into RNase-free water (Which is a type of
nuclease-free water), TE buffer and specialized RNA dissolving
solution. Commercially available RNA dissolving agents also
contain RNA stabilizing agents which is more advisable.
 Take a look at some recommendations for dissolving RNA.
 Dissolve RNA in RNase-free water or RNase-free pre-autoclave
TE buffer.
 Do not dry the RNA pellets by heat.
 Store dissolved RNA at -80℃.
 Use RNase inactivation solution to store the RNA.
 Do not store it with other tissue or DNA samples.
 RNA quality checks:
 The conventional agarose gel electrophoresis technique is used to
determine the presence of RNA and investigate the quality of
RNA. The total extracted RNA is run on a 0.8 to 1% agarose gel.
Difference Between Plant and Animal DNA Extraction :
The key difference between plant and animal DNA extraction
is that in plant DNA extraction, it is necessary to break the
cell wall by grinding the tissue in dry ice or liquid nitrogen in
order to release cellular contents while in animal DNA
extraction, it is not necessary to carry out this step since animal
cells do not have a cell wall.
Genomic DNA is the genetic material of plant and animal
cells. Genomic DNA is unique to each individual plant or
animal. The DNA extraction of good-quality DNA is the
prerequisite for molecular research.
DNA of organisms is extracted for identification, diagnosis of
diseases, detection of specific genes and sequences, forensic
purposes, paternity testing, genome sequencing, and
development of drugs, etc. DNA extraction method varies
depending on the type of cells – whether it is
an animal cell or a plant cell.