Manual Extraction of DNA from The Blood. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.- Materials

Manual Extraction of DNA from The Blood

-Blood Sample.

-Distilled water.

Dionized water. -

Ice and Plastic bucket.-

Materials

Auto clave.- PH meter.-

Balance.- -Micro pipette (transfer pipette) (2-20µl, 10-100µl,

100-1000µl) Centrifuge.-

Spectrophotometer.-

Equipment

-Beakers Volumetric flask -

-Plastic and glass centrifuge tubes -Measuring cylinders.

-Pasteur pipettes.- -Pipettes (1ml, 5ml, 10ml) .

-Eppendorf tube. -Tips with different size.

Racks.- Quarts cuvettes.- 50ml 15ml

Glassware

1M Tris PH= 7.6 Lysis buffer (2X).-

SDS 10%.- Salt/ EDTA.-

EDTA 0.5M .- Ethanol 99-100%.-

10mM Tris, 1mM EDTA.- 1M Tris PH= 8-

- TE buffer 10:1

Reagent

Add 300µl of blood + 900µl of tris-cl pH = 7.6 (Lysis buffer(2X) ) to an eppendorf tube to lyse red blood

cells .

Mix the samples and incubate it at room temp. for 1 min

Put the tubes in the Centrifuge for 20 sec at maximum speed.

There will be 2 layers

Extraction and purification of DNA Procedure

Discard the supernatant.

Add 200 µl of tris-cl pH = 8 + 200 µl EDTA buffer + 200 µl of 10%SDS to the pellet.to lyse WBS

Put the tubes in the centrifuge for 5 min at max. speed



Take the supernatant in another clean eppendorf

Add 60 µl .potassium acetate glecial to precipitate protein

Put the tubes in the Centrifuge for 10min at 2000rpm.

There will be tow layers.



Add 600 µl of ethanol to each tube to precipitate the DNA.

Put the tubes in the Centrifuge for 10min at 2000rpm.

Discard supernatent



Put the tubes up side down until the precipitated DNA is completely dry.

Add 100 µl of TE buffer in the eppendorf tube to redissolve the DNA over night .



Dilute 25µl of DNA sample with 2ml of distilled water in quartz cuvette and mix throughly.

The concentration of DNA sample was assessed by using spectrophotometer.

The optical density was recorded at 260 and 280nm .

The 260/280nm absorbance ratio was calculated.

Measure of DNA concentration

Any Question?

Thank You

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Manual Extraction of DNA from The Blood. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.- Materials