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Manual Dna Extraction From Blood Through Salting Out Procedure

This document provides a procedure for extracting DNA from human blood samples through a salting out method. The salting out procedure allows for rapid and safe DNA extraction without the need for expensive or hazardous reagents like phenol and chloroform. The procedure involves lysing blood cells, digesting the cell lysates with SDS and proteinase K, adding saturated NaCl to precipitate proteins, collecting the supernatant containing DNA, precipitating the DNA with ethanol, and dissolving the extracted DNA in TE buffer.

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Abner Piedra
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32 views1 page

Manual Dna Extraction From Blood Through Salting Out Procedure

This document provides a procedure for extracting DNA from human blood samples through a salting out method. The salting out procedure allows for rapid and safe DNA extraction without the need for expensive or hazardous reagents like phenol and chloroform. The procedure involves lysing blood cells, digesting the cell lysates with SDS and proteinase K, adding saturated NaCl to precipitate proteins, collecting the supernatant containing DNA, precipitating the DNA with ethanol, and dissolving the extracted DNA in TE buffer.

Uploaded by

Abner Piedra
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOC, PDF, TXT or read online on Scribd
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Gnthon

Fodor Jzsef National Center for Public Health

Laboratory Procedures for Human DNA Extraction


December 2014

MANUAL DNA EXTRACTION FROM BLOOD through


SALTING OUT PROCEDURE

One of the obstacles encountered when extracting DNA from a large number of samples
is the cumbersome method of DNA extraction with the organic solvents phenol and
chloroform.
This method is rapid, safe and does not require expensive and environmentally
hazardous reagents and equipment.

Equipment and Materials

Polypropylene tubes 15ml


Lysis buffer (10mM Tris-HCL,400mM NaCl, 2mM Na2EDTA, pH 8.2)
SDS 10%
Proteinase K solution (1 mg proteinase K in 1% SDS and 2 mM Na2 EDTA).
Centrifuge
Absolute ethanol
TE buffer (10mM Tris-HCL, 0.2mM Na2 EDTA, pH 7.5)
Disposable gloves
Gilson pipette

Procedure
1. Resuspend the buffy coats of nucleated cells obtained from blood with
anticoagulents (ACD or EDTA) with 3ml of nuclear lysis buffer.
2. Digest the cell lysates, with 0.2 ml of 10% SDS and 0.5 ml of proteinase K
solution, overnight at 37 C.
3. Add 1ml of saturated NaCl (6M) to each tube and shake vigorously for 15 seconds.
4. Centrifuge for 15 minutes at 2500 rpm.
5. Transfer the supernatant containing the DNA to another 15ml polypropylene tube,
the precipitated protein pellet is left behind at the bottom of the tube.
6. Add 2 volumes of absolute ethanol and invert the tubes several times until the
DNA precipitates.
7. Remove the precipitated DNA with a plastic spatula or pipette and transfer to a
1.5ml microcentrifuge tube containing 100-200 microliter TE buffer
8. Dissolve the DNA for 2 hours at 37C
9. Store the tube at +4 or 20C.
10. Check quantity/quality of DNA (see QUALITY CONTROL OF DNA protocol)

Reference
Miller S.A, Dykes D.D, Polesky H.F : A simple salting out procedure for extracting DNA
from human nucleated cells. Nucleic Acids Research 1988; V16 Number 3 : 1215

Gnthon-S.Saker, NCPH-V.Karcagi
Copyright Eurobiobank 2014

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