Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
2. There are five basic steps of DNA extraction that
are consistent across all the possible DNA
purification chemistries:
1)disruption of the cellular structure to create a lysate,
2) separation of the soluble DNA from cell debris and
other insoluble material,
3) binding the DNA of interest to a purification matrix,
4)washing proteins and other contaminants away
from the matrix and
5)elution of the DNA.
Nucleic acid purification
3. Electro elution of DNA from agarose gel
Isolated DNA sample mix with gel loading buffer and load it into slot in 0.8% agarose gel
containing ethidium bromide.
Carry out the electrophoresis until the tacking dye reach to end of gel
Observed gel under UV trans-illuminator and cut out the eluted DNA band with blade
Transfer the gel DNA band into the dialysis bag and add the buffer into dialysis bag until DNA
block immerse into buffer
Place dialysis tube in electrophoresis apparatus and pour enough working buffer
Submerge the bag completely in the working buffer then apply the current 30mA for 1and half
hr.
Remove the dialysis bag from apparatus and cut off bag at one end
Transfer dialyzed DNA buffer into centrifuge tube and add phenol and chloroform- isoamyl
alcohol and mix it well
centrifuge tube at 4000rpm for 10min and transfer upper layer to new tube
Add sodium acetate and twice volume of ethanol and leave it in deep freezer at -20oC
Centrifuge at 10000rpm for 20min discard the supernatant and dry the pellet
Add the ethanol in pellet and spin at 12000rpm for 30min and then dry pellet under vacuum
Re-Suspended the pellet in D/W for further use.
4.
5. Biogel column purification of DNA
Fix the column to the stand and pack it with glass wool at lower
end
Mix the DEAE-cellulose in the column buffer and allow the gel
swell for 10min.
Transfer the swollen gel into column and allow the gel settle in
column for 10min.
Wash column with column buffer 3-4times then use it
Add the DNA solution at the top of the column and collect the elute
and discard.wash the column with column buffer 3-4 times.
Add elution buffer and collect elution solution which contain the
DNA
Add little sterile D/W to eluted DNA to dilute the conc.NACl
Precipitate the DNA with add the ethanol and centrifuge the
precipitate at 12000rpmfor 30min
Dry pellet and re suspended into D/W for further use
6. Density gradient Centrifugation :A procedure for separating
particles (such as viruses or ribosomes or molecules such as DNA
)in which the sample is placed on a preformed gradient such as
sucrose or caesium chloride. Upon centrifugation either by rate
zonal or equilibrium procedures, the macromolecules are 'banded'
in the gradient and can be collected as a pure fraction.
Density gradient centrifugation are of two types:
1. Rate zonal centrifugation
2. Isopycnic centrifugation
Density gradient Centrifugation
7. Density gradient centrifugation of DNA
Take the DNA sample solution centrifuge it at 13000rpm for 30min and add the
cesium chloride(CS) and ethidium bromide
Then again spin the tube in centrifuge at 35000 to 40000rpm for 2-3 days at10-
120C.Take out the tube and observed DNA bands with UV-trans illuminator
Fox tube with a clamp on vertical stand for elution of band and puncture the
tube through tape at lower edge of DNA band with sterile syringe
Run off fluorescent DNA band material into small dry tube. And separate
the ethidium bromide from DNA material with isopropanol
Add equal volume of CsCl solution in isopropanol to tube and shake gently.
The ethidium bromide will come out in isopropanol will turn into pink
solution.
Remove the pink colored solution , these step repeat until remove all pink
stain. And the remove CsCl transfer DNA sample in dialysis bag then open one
end of bag
Place the bag in a beaker and pour the TE buffer to it and dialyze overnight, then
transfer the DNA from Dialysis tube to bottle and store at 40C
8. Isolated DNA cut with RE EcoRI . After
cutting DNA sample heat at 65OC for
10min.
Dilute the sample with TE buffer for
reduced the Mg++ conc.
Add NACl and PEG in these solutionand
dissolve it and incubate at 37OC for 20Hr.
Centrifuge sample at 10000rpm for
10min and collect the supernatant
Add the more PEG for final conc.18%
and dissolve it and incubate the sample
for 2-3 hr at 00C
Precipitated DNA centrifuge at
10000rpm at 4 0C. And pellet pf DNA re
suspended in TE buffer.
Precipitate the DNA in ethanol and
resuspended in suitable buffer
Purification of DNA with PEG
