Protocol - DNA extraction from whole blood
DNA extraction from whole blood using a salting out procedure
Category:
Molecular Biology
Creation date: Feb 05, 2000
Last revision: Dec 19, 2014
Contact
Name: Giuseppina Lacerra
Address: IGB-CNR via Pietro Castellino 111, 80131 Napoli
Steps
| Description | Temperature | Time | Note |
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Collect 10 ml of peripheral blood into tubes containing EDTA or citrate.
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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.
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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
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R.T.
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1-2 min
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This steps have be done at room temperature to avoid the hemolysis of blood.
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Centrifuge at 2,500 rpm
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R.T.
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10 min
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Remove and discard supernatant and repeat the step 2-4 more times, or until no hemoglobin remains.
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R.T.
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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.
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4°C
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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-
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Close the tube and shake the resulting mixture well by hand. Keep the samples on ice
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4° C
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5 min
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Centrifuge at 4.000 rpm.
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4° C
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10 min
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Repeat the steps of hemolysis with TE 1X until the solution is clear.
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4° C
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Proteinase digestion: resupend the pellet in 4,5 ml of TE 2X. Shake the pellet well by vortex.
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4° C
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Add 0.5 ml of SDS 10% and 300 ul of Proteinase K 10 mg/ml. Shake gently.
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Incubate
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37°C
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over night
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The incubation can be performed also al 65°C for 1 hour.
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Add 1,6 ml of NaCl solution (5.5 M). Shake vigorusly by vortex.
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Centrifuge at 5,000 rpm
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R.T.
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15 min
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Following centrifugation at the bottom of the tube there will be a pellet containg the proteins with the salt.
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DNA precipitation: Transfer the aqueous phase to a fesh tube and add an equal volume (about 4.5 ml) of isopropilic alcool.
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Shake gently by inveting the tube.
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Centrifuge at 5.000 rpm
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R.T.
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10 min
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DNA wash: Remove the supernatant leaving some ul above the pellet and wash the DNA pellet with 1 ml of 70% ethanol.
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Centrifuge at 5.000 rpm
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4° C
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10 min
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DNA solubilization: Remove and discard the ethanol and briefly air-dry the DNA pellet
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R.T.
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10 min
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Dissolve the DNA in about 0.5 ml of TE 1X.
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Store the tube in a refrigerator
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4° C
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over night
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Transfer the DNA in a 1.5 ml fresh tube.
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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.
