DNA is extracted from human Blood cells
for a variety of reasons
Forensics:
Evolution:
Medical:
Bio-engineering or cloning
General research purposes
Before DNA Extraction
Take blood (5ml-10ml) via sterile syringe
Pore blood in to anticoagulant tube or a falcon tube
containing 200 micro liter 1M EDTA
Store at -80 for 4,5 hours or -20 for 1 day to provide cold
stress to RBC,s
Thaw blood properly before extraction and mix gently
Reagents required
Lysis buffer (10mM Tris HCL, 2mMEDTA, pH 8.0)
TNE buffer (Tris HCL 10 mM, EDTA 2mM, NaCl 400mM)
10% SDS
Proteinase-K solution 20mg/ml
6M NaCl
Phenol-Choloroform-Isoamylalcohol (PCI) (25:24:1)
Chilled Isopropanol
75% Ethanol
Low TE buffer (10mM Tris, 0.2mM EDTA)
Major Steps
Cell lysis and purification of
leukocytes
Proteins and Lipid degradation
DNA purification
DNA Extraction (1 day) st
Before starting the DNA extraction liquefy or thaw the
samples of blood and take 250ul (micro liter)Blood
from stock.
Add up to 1 ml of lysis buffer in 1 ml blood containing
Eppendorf.
Centrifuge at 6000 rpm for 10 minutes ( All
centrifugation in successive phases should be done at
8C)
Remove 250 ul supernatant. Breakdown the pellet
made at lowermost by taping it gradually. Add lysis
buffer up to 1 ml.
Centrifuge at 6000 rpm for 10 minutes ( All
centrifugation in successive phases should be done
at 8C)
Remove 500 ul supernatant. Breakdown the pellet
made at lowermost by taping it gradually. Add lysis
buffer up to 1 ml.
Centrifuge at 6000 rpm for 10 minutes ( All
centrifugation in successive phases should be done
at 8C)
Remove 750 ul supernatant. Breakdown the pellet
made at lowermost by taping it gradually. Add lysis
buffer up to 1 ml.
Centrifuge at 6000 rpm for 10 minutes ( All
centrifugation in successive phases should be done at
8C)
Remove 900 ul supernatant. Breakdown the pellet
made at lowermost by taping it gradually. Add lysis
buffer up to 1 ml.
Centrifuge at 6000 rpm for 10 minutes ( All
centrifugation in successive phases should be done at
8C)
Remove all supernatant. Breakdown the pellet
made at lowermost by taping it gradually. Add lysis
buffer up to 1 ml.
Centrifuge at 6000 rpm for 10 minutes ( All
centrifugation in successive phases should be done
at 8C)
Remove the supernatant leaving pellet and re-
suspend pellet in 150 ul TNE buffer for 250 micro
liter initial blood volume. Add 20 ul 10% SDS and
2ul Proteinase K.
Samples were incubated overnight in 37dnnn C
shakers
Day 2 nd
Whole digestion of the pellet is crisscross after one
night incubation. If the pellet is not fully digested
then add additional Proteinase K according to the
quantity of undigested pellet. Once more incubated
at 37C for 2-3 hours or till the pellet is fully
digested.
Inorganic DNA Extraction Method:1st day protocol is
same in both methods:
We preferred inorganic DNA Extraction protocol for
fresh samples.
The tubes should kept on snow and added 150 ul
saturated NaCl (6 M) for 250 ul initial blood volume. The
falcon tubes were shaken energetically and place on ice
for a second time for 10-15 minutes.
Centrifuged at 6000 rpm for 10 minutes to settle down
the pellet (salts and proteins).
Supernatant poured in a labeled Eppendorf.
Centrifuged at 6000 rpm for 10 minutes
Organic DNA Extraction Method by Phenol, Chloroform,
Isoamylalcohol (PCI):
For old samples we applied Organic DNA Extraction method (PCI).
1st day protocol is same in both methods:
Phenol : Chloroform : Isoamylalcohol
25 : 24 : 1
Add 150 ul of PCI for 250 ul initial blood volume. The falcon tubes
were shaken energetically and place on room temperature for
15-20 minutes.
centrifuged at 6000 rpm for 10 minutes to settle down the pellet
(salts and proteins).
The supernatant poured in a labeled Eppendorf.
Centrifuged at 6000 rpm for 10 minutes
The same amounts of Isopropanol were added and
overturned the tubes moderately until DNA was
obvious.
The tubes were leaved for 5 minutes on ice.
Again Centrifuged the samples at 6000 rpm for 10
minutes
The supernatant were thrown away carefully
The DNA pellet was wash away with 150 ul of 70 %
ethanol for 250 ul initial blood volume.
Centrifuged again at 6000 rpm for 10 minutes
70 percent ethanol was removed wisely leaving
the pellet.
The DNA pellet was air dried out in 37C air dryer.
Added 40 ul low T.E (Tris HCL 10mM, EDTA
0.2mM)/ or in Injection Water
The tubes were placed in 37C shaking incubator
overnight to melt the DNA. Covered bands of Para
film round the tip of the tubes.
3rd day
Keep the tubes in a shaky water bath 70C for an hour
to deactivate nucleases
The tubes were left at room temperature to be chilled
Samples were Spin briefly
2ml autoclaved tubes were labeled by side and cap.
Tubes were labeled including pedigree number
individuals ID.
DNA samples were Aliquoted in duplication and stored
at -20C conferring to pedigree number in marked and
numbered Cryoboxes.
The samples were Stored at -80 C for long term
storage
