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Dna Extraction From Blood: Preparation of Buffers

The white blood cells in peripheral blood provide a convenient source of human genomic DNA for analysis of hemoglobinopathies. Approximately 10 ml of whole blood yields around 250 μg of DNA, sufficient for analysis of globin genes using PCR. DNA extraction uses buffers containing EDTA to inhibit nucleases, enzymes that break down nucleic acids, since DNA is vulnerable to degradation when cells are lysed and their membranes disrupted. The procedure involves lysing blood cells, isolating the nuclei, further lysing the nuclei, precipitating the DNA from solution using ethanol, and resuspending the purified DNA in buffer for storage.

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142 views2 pages

Dna Extraction From Blood: Preparation of Buffers

The white blood cells in peripheral blood provide a convenient source of human genomic DNA for analysis of hemoglobinopathies. Approximately 10 ml of whole blood yields around 250 μg of DNA, sufficient for analysis of globin genes using PCR. DNA extraction uses buffers containing EDTA to inhibit nucleases, enzymes that break down nucleic acids, since DNA is vulnerable to degradation when cells are lysed and their membranes disrupted. The procedure involves lysing blood cells, isolating the nuclei, further lysing the nuclei, precipitating the DNA from solution using ethanol, and resuspending the purified DNA in buffer for storage.

Uploaded by

hely shah
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA EXTRACTION FROM BLOOD

The white blood cells (WBC) of peripheral blood are usually the most convenient source
of human genomic DNA for DNA analysis with respect to haemoglobinopathies. It is estimated
that 10 ml of whole blood yield approximately 250 μg of DNA, more than sufficient for
complete analysis of globin genes with the methods that are currently available (ie based on
PCR). DNA is an extremely stable molecule, but enzymes which catalyse the breakdown of
nucleic acids (nucleases) are found in all cells. In intact cells the DNA is found in the nucleus
and thus is protected from the action of nucleases which are abundant in the lysosomes in the
cytoplasm. However when cells are lysed, the membranes of the cell compartments are
disrupted, allowing nucleases to come in to contact with the DNA. Thus DNA extraction uses
buffers which contain inhibitors of nuclease activity i.e: (EDTA)
Preparation of buffers

Cell lysis buffer (CLB)

The final volume will be 1000ml that contains 100mM Tris-Hcl (pH 7.6), 5mM MgCl 2, 7mM
KCL, 300mM sucrose and 1% Triton X-100.

Nucleus Lysis Buffer (NLB)

The nucleus lysis buffer contains 100mM Tris-Hcl, 12mM sodium acetate/ potassium acetate or
10mM sodium citrate, 2% sodium dodecyl sulphate (SDS) and 10mM EDTA.

DNA Suspending Buffer (TE buffer)

10mM Tris-Hcl and 1mM EDTA. For 1 litter

5m sodium chloride (NaCl) solution

Dissolve 29.2 gm of NaCl in 100ml distill water.

All the buffer must be sterilized prior use and before starting DNA extraction, chloroform and
o
absolute ethanol (100%) should be kept on -20 C before starting DNA extraction.

PROCEDURE

i. Take 300 ul of blood in 1.5 ml microcentrifuge (Ependroff) tube and add 500 ul of CLB
in it.
ii. Mix it by inversion and keep the tubes at room temp for 5 min
iii. Centrifuge tube for 5 min at 5000 rpm at 25 C. and then discard supernatant
iv. Again add 400 ul of CLB in pallet and centrifuge it for 5 min at 5000 rpm. Repeat
this procedure 3-5 times until get a clear white pallet. ( NUCLEAR PALLETE)
v. Discard the supernatant and add 400 ul of Nuclear Lysis buffer, then 100ul of 5M NaCl
solution.
vi. Dissolve pallet and add 550ul of chilled chloroform
vii. Leave the tube for 10 min then centrifugation at 5000rpm for 3minute. Two separate
layers will be formed. Carefully withdraw the supernatant without touching the central
layer to new Eppendorf tube
o
viii. Add 1000µl pre-chilled ethanol and centrifuge at maximum speed for 6min at 4 C
to precipitate DNA
ix. Pour off ethanol slowly and washed the DNA with 70% ethanol, centrifuge at maximum
for 6 minutes.
x. Decant the ethanol and left the tube at room temperature until completely dry.
o o
xi. Add 70µl TE buffer, vertex for 10 seconds and store the DNA at 4 C and/or -20 C.

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