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Protocol - DNA extraction from whole blood

DNA extraction from whole blood using a salting out procedure

CategoryMolecular Biology
Creation dateFeb 05, 2000
Last revisionDec 19, 2014
NameGiuseppina Lacerra
AddressIGB-CNR via Pietro Castellino 111, 80131 Napoli


Description Temperature Time Note
Collect 10 ml of peripheral blood into tubes containing EDTA or citrate.
The extraction of DNA can be done from fresh or frozen blood. The blood wash steps have to be done only for the extraction from fresh blood.
Fresh Blood wash: Transfer the blood sample in 50 ml Falcon and add saline solution (0.90% w/v of Na Cl) up to a volume of 50 ml. Shake gently
1-2 min
This steps have be done at room temperature to avoid the hemolysis of blood.
Centrifuge at 2,500 rpm
10 min
Remove and discard supernatant and repeat the step 2-4 more times, or until no hemoglobin remains.
Blood lysis: Remove and discard the supernatatnt and add TE 1X up to 50 ml. Shake the resulting mixture well by hand. Keep the samples on ice.
Starting from this step,, it is important to put the samples in ice and to use refrigerated solution, to inibit the DNasi activities. Strat from this point if you use frozen blood-
Close the tube and shake the resulting mixture well by hand. Keep the samples on ice
4° C
5 min
Centrifuge at 4.000 rpm.
4° C
10 min
Repeat the steps of hemolysis with TE 1X until the solution is clear.
4° C
Proteinase digestion: resupend the pellet in 4,5 ml of TE 2X. Shake the pellet well by vortex.
4° C
Add 0.5 ml of SDS 10% and 300 ul of Proteinase K 10 mg/ml. Shake gently.
over night
The incubation can be performed also al 65°C for 1 hour.
Add 1,6 ml of NaCl solution (5.5 M). Shake vigorusly by vortex.
Centrifuge at 5,000 rpm
15 min
Following centrifugation at the bottom of the tube there will be a pellet containg the proteins with the salt.
DNA precipitation: Transfer the aqueous phase to a fesh tube and add an equal volume (about 4.5 ml) of isopropilic alcool.
Shake gently by inveting the tube.
Centrifuge at 5.000 rpm
10 min
DNA wash: Remove the supernatant leaving some ul above the pellet and wash the DNA pellet with 1 ml of 70% ethanol.
Centrifuge at 5.000 rpm
4° C
10 min
DNA solubilization: Remove and discard the ethanol and briefly air-dry the DNA pellet
10 min
Dissolve the DNA in about 0.5 ml of TE 1X.
Store the tube in a refrigerator
4° C
over night
Transfer the DNA in a 1.5 ml fresh tube.

Other informations

For more info see the paper:  A simple salting out procedure for extracting DNA from human nucleated cells.  Miller SA, Dykes DD, Polesky HF. Nucleic Acids Res. 1988 Feb 11;16(3):1215.


Quality validation: Yes

Validation info

We performed several salting out extraction and compared the spectrophotometrich parameters with that obtained with the phenol clorophorms protocols.

The protocol has been extensively used to produce data published in peer-reviewed journals.


Lacerra G, Scarano C, Lagona LF, Testa R, Caruso DG, Medulla E, Friscia MG, Mastrullo L, Caldora M, Prezioso R, Gaudiano C, Magnano C, Romeo MA, Musollino G, Di Noce F, Carestia C. Genotype-phenotype relationship of the δ-thalassemia and Hb A(2) variants: observation of 52 genotypes. Hemoglobin. 2010;34:407-23.
Lacerra G, Scarano C, Musollino G, Testa R, Prezioso R, Caruso DG, Lagona LF, Medulla E, Friscia MG, Gaudiano C, Carestia C. HbA2-Partinico or delta(A2)Pro-->Thr, a new genetic variation in the delta-globin gene in cis to the beta(+) thal IVS-I-110 G>A, and the heterogeneity of delta-globin alleles in double heterozygotes for beta- and delta-globin gene defects. Ann Hematol. 2010;89:127-34.
Lacerra G, Musollino G, Scarano C, Lagona LF, Caruso DG, Testa R, Prezioso R, Di Noce F, Medulla E, Friscia MG, Mastrullo L, Caldora M, Nota L, Gaudiano C, Magnano C, Ciaccio C, Romeo MA, Carestia C. Molecular evidences of single mutational events followed by recurrent crossing-overs in the common delta-globin alleles in the Mediterranean area. Gene. 2008;410:129-38.
Lacerra G, Fiorito M, Musollino G, Di Noce F, Esposito M, Nigro V, Gaudiano C, Carestia C. Sequence variations of the alpha-globin genes: scanning of high CG content genes with DHPLC and DG-DGGE. Hum Mutat. 2004;24:338-49.
De Angioletti M, Lacerra G, Gaudiano C, Mastrolonardo G, Pagano L, Mastrullo L, Masciandaro S, Carestia C. Epidemiology of the delta globin alleles in southern Italy shows complex molecular, genetic, and phenotypic features. Hum Mutat. 2002;20:358-67.


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