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The DNA Extraction Process Frees DNA From The Cell and Then Separates It From Cellular Fluid and Proteins So You Are Left With Pure DNA

The DNA extraction process involves several steps to free DNA from cells and separate it from other cellular components. This leaves pure DNA. The key steps include extracting whole blood, separating white blood cells using EDTA and centrifugation, adding NaCl and SDS to lyse cells, adding RNase and phenol:chloroform:isoamyl alcohol to separate DNA from other materials, and precipitating DNA from the aqueous phase using isopropyl alcohol before washing with ethanol and dissolving in Tris EDTA solution.

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51 views2 pages

The DNA Extraction Process Frees DNA From The Cell and Then Separates It From Cellular Fluid and Proteins So You Are Left With Pure DNA

The DNA extraction process involves several steps to free DNA from cells and separate it from other cellular components. This leaves pure DNA. The key steps include extracting whole blood, separating white blood cells using EDTA and centrifugation, adding NaCl and SDS to lyse cells, adding RNase and phenol:chloroform:isoamyl alcohol to separate DNA from other materials, and precipitating DNA from the aqueous phase using isopropyl alcohol before washing with ethanol and dissolving in Tris EDTA solution.

Uploaded by

Nisar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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COMDNA EXTRACTION

The DNA extraction process frees DNA from the cell and then separates it from cellular fluid and
proteins so you are left with pure DNA.
1. Extract whole blood
2. Separate wbc from it by using hypotonic EDTA
3. Then centrifuge the solution,  pellet contains WBCs
4. Step 3 can be repeated for better results
5. Add a small amount of NaCl solution to the pellet
6. Add SDS to the solution
7. Add RNase
8. Add Phenol:chloroform:isoamyl alcohol (PCI)
9. DNA precipitates in aqueous phase with isopropyl alcohol
10. Wash it with ethanol and then dissolve in Tris EDTA Solution
The sample can be quantified, stored and also used!

Before starting DNA extraction, liquid blood venogects should be shake gently by
rotating blood mixer (vortex)
1. Pour 500 μl of blood into a 1.5 ml eppendorf tube and add 1000 μl of red cell lysis buffer.
2. Shake microfuge tube gently (up to homogenizing), then spin for 2 minutes at 7000 rpm.
3. Discard supernatant and repeat steps 1-3 two or three more times to remove hemoglobin. It is
important to breakdown the pellet by vortexing and rinses it well in red blood
cell lysis buffer in order to clean the white blood cells from residual of hemoglobin.
4. Placing the tube on tissue paper for few seconds downward. Be careful from cross-
contamination between different samples.
5. Add 400 μl of nucleic lysis buffer to eppendorf tube. Note: if the pellet formed, you must
pipette the pellet up to dissolve it.
6. Add 100 μl of saturated NaCl (5M) and 600 μl of chloroform to eppendorf tube and mix on a
rotating blood mixer at room temperature then spin it for 2 minutes at 7000
rpm.
7. Transfer 400 μl of supernatant to a new 1.5 ml tube.
8. Add 800 μl of cold (-20°C) absolute Ethanol and shake it gently then vortex it. DNA should
appear as a mucus-like strand in the solution phase.
9. Spin the microfuge tube for one minute at 12000 rpm to precipitate, then discard supernatant
carefully and let tube be completely dried in room temperature (Place
Eppendorf tube downward on the tissue paper).
10. Add 50μl of TE to it then vortex; keep eppendorf tube of DNA in 4°C or -20°C for later uses.
We routinely use about one μl per PCR reaction without adverse affects. DNA
can be quantified and diluted to a working concentration at this point or simply use 1 μl per PCR
reaction.

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