Day :
Wednesday
Date :
21/11/2012
Lab : Genetics - Clinical Rotation
Exp. Title:
by Silica Based method
(Qiagen)
Background
The genetic material of all living organisms is DNA, which located in the nucleus of
Eukaryotic cell & in the cytoplasm of Prokaryotic cells. If any change occurred to DNA sequence (due to mutations) this may cause a genetic disease. To study a genetic disease we Have to know the abnormal one (patient sample) to be compared with the normal one.
To study the sequence of any gene we have to extract the whole genome from the patients sample which could be any of the following :
Blood, Serum & Plasma, Semen, Saliva, Urine, Hair (w/Root & Shaft), Teeth,
Bone, Tissue, Cigarette Butts, Envelope & Stamps, Fingernail Clippings,
Chewing Gum, Bite Marks, & Feces.
In Today's experiment we will perform the DNA extraction from blood samples, that mean we will extract the DNA from the nucleus of WBCs. As long as the blood contains other cells and substances we have to get rid of them.
The DNA extract should be pure so we have to get rid of :
1.
RBCs 2. Platelets 3. Plasma proteins
4. WBCS components including : plasma membrane , cytoplasmic & nuclear proteins, nuclear membrane, chromatin (like histones).

1.
Organic method
 Uses organic solvents' (phenol:chloroform)
 Advantages : very basic and easy to perform
 Disadvantages : Toxic organic solvents, need multiple step of washing which increase the risk of contamination, need a long time to be done (overnight incubation)
2.
Non organic method ( Salting out method)
 Using strong basis like NaOH
 It is difficult to remove the salts from the DNA billet, and that will affect the purity of
DNA
3.
Anion exchange method
4.
Magnetic method
5.
Silica gel – membrane spin method 
 Advantages over other methods : easy to perform, fast, good yield of DNA, good purity, no need for toxic organic solutions, decreased risk of contamination, because it includes only 2 fast steps of washing.
 Disadvantages : multiple sample transfer , which may lead to contamination
Principle:
Spin column procedures is simple. Samples are first lysed using proteinase K. Buffering conditions are adjusted to provide optimal DNA binding conditions and the lysate is loaded onto the Mini spin column. During centrifugation, DNA is selectively bound to the membrane as contaminants pass through. Remaining contaminants and enzyme inhibitors are removed in two efficient wash steps and DNA is then eluted in water or buffer, ready for use.
(Selective adsorption of DNA to silica-gel membranes under controlled ionic conditions )

The silica gel – membrane is composed of a porous substance with a positively charged material called (DEAE-
Diethylaminoethanol), which can react with the negatively charged phosphate groups of the DNA backbone and this lead to DNA entrapment and separation.
Blood Sample :
Any anticoagulated blood : EDTA, Heparin, or Cetrate spin column
Mini
Equipments :

Pipets and pipet tips

Vortexer

Microcentrifuge tubes (1.5 ml or 2 ml)

Microcentrifuge with rotor for 1.5 ml and 2 ml tubes

Thermomixer, shaking water bath, or rocking pla tform for heating at 56°C

Mini spin columns
Reagents :

AW1 buffer : washing buffer -1

AW2 buffer : washing buffer -2

AE buffer : Elution buffer

Tris.HCl, EDTA, pH 9.0

AL buffer : Lyses buffer  lyses cell membrane & nuclear membrane

Tris.HCl : pH7.5
 Sucrose : make hypotonic solution, this lead to cell rupture & increase nuclei
density

Triton-X100 : disrupt cell membrane & nuclear membrane
 EDTA : chelates Mg+, this lead to inactivate Nucleases enzymes (DNase, &
RNase)

Proteinase – K : digest proteins

Absolute Ethanol 96-100% : remove water from DNA & precipitate it

Procedure :
1.
Pipet 20 μl proteinase K into a 1.5 ml or 2 ml microcentrifuge tube.
2. Add 2
00 μl anticoagulated blood.
3. Add 200 μl Buffer AL . Mix thoroughly by vortexing,  incubate at 56°C for 10 min.
4. Add 200 μl absolute ethanol (96–100%) to the sample, and mix thoroughly by vortexing.
5. Place a DNeasy mini spin column in a 2 ml collection tube
6. Pipet the mixture from step 4 into the DNeasy Mini spin column. Centrifuge at (8000 rpm) for 2 min.
7. Discard the collection tube.
8.
Place the DNeasy Mini spin column in a new 2 ml collection tube, & add 500 μl
Buffer AW1, and centrifuge for 2 min at (8000 rpm).
9. Discard collection tube.
10. Place the DNeasy Mini spin column in a new 2 ml collection tube & add 500 μl Buffer
AW2, and centrifuge for 3 min at(14,000 rpm) to dry the DNeasy membrane.
11. Discard the collection tube.
12. Place the DNeasy Mini spin column in a clean 2 ml microcentrifuge tube, and pipet
200 μl Buffer AE directly onto the DNeasy membrane. Then Incubate at room temperature for 1 min, and then centrifuge for 2 min at (8000 rpm) to elute the DNA.
13. Store the DNA at -20 degree in the freezer.