What method did you use?

You've made a mistake somewhere in your protocol

If you have the resources, there are many commercial kits available that make DNA isolation from blood a breeze. I use the Qiagen DNA Blood Mini Kit. I use only 100 uL of blood and get very pure and concentrated DNA. If you don't want to use kits, perhaps try isolating DNA after isolating PBMC from the whole blood. You'll get a higher concentration of DNA and won't have to worry about red blood cells.

Hi Muhammad,

What are you using the DNA for....??? If its for a PCR based assay, then maybe you should save yourself the trouble, and PCR directly from blood.....there are several kits currently available.

I have had great success with these, using as little as 1-2microlitres of fresh (or frozen)-EDTA blood......

Salaam: You did not probably separate proteins 100%. I suggest go to the phase separation step (Phenol-chloroform step in case of manual method) where DNA and protein are separated and repeat the process. Let me know if it works.

You probably need to shake the tube to mix the isopropanol with the nucleic acid contained in the aqueous phase. Then let sit for a few minutes at room temp or colder. Then centrifuge at high speed for 20-30 minutes. You should see a pellet at the bottom of the tube that is your nucleic acid. Here is a detailed description from this website:

http://www.qiagen.com/knowledge-and-support/faq/?ID=5cf3006a-c63f-48d6-89d9-9ae14329c230

How can I precipitate genomic DNA using isopropanol?

FAQ ID -2953

Alcohol precipitation is commonly used for concentrating, desalting, and recovering nucleic acids. Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitation of DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co-precipitation of salt that interferes with downstream applications.

Procedure

Adjust the salt concentration, for example, with sodium acetate (0.3 M, pH 5.2, final concentration) or ammonium acetate (2.0–2.5 M, final concentration).

Add 0.6–0.7 volumes of room-temperature isopropanol to the DNA solution and mix well.

Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C

Carefully decant the supernatant without disturbing the pellet.

Wash the DNA pellet by adding 1–10 ml (depending on the size of the preparation) of room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

Centrifuge at 10,000–15,000 x g for 5–15 min at 4°C.

Carefully decant the supernatant without disturbing the pellet.

Air-dry the pellet for 5–20 min (depending on the size of the pellet).

Redissolve the DNA in a suitable buffer.

Tip: Use a buffer with a pH of 7.5–8.0, as DNA does not dissolve easily in acidic buffers. Often distilled water can have an acidic pH. The addition of EDTA protects the DNA from DNase digestion.

Tip: High-molecular-weight DNA, such as genomic DNA, should be redissolved very gently to avoid shearing. If the DNA pellet does not dissolve easily, heat at 55°C for 1–2 h with gentle shaking.