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Manual Extraction of DNA from The Blood. Prepared by: Kholoud Al- Homoudi Rana Al-Turki . Materials. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket. -. Equipment. Auto clave. - PH meter. - Balance. -

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manual extraction of dna from the blood

Manual Extraction of DNA from The Blood

Prepared by: Kholoud Al- Homoudi

Rana Al-Turki

slide2

Materials

- Blood Sample.

- Distilled water.

Dionized water. -

Ice and Plastic bucket.-

slide3

Equipment

Auto clave.-

PH meter.-

Balance.-

- Micro pipette (transfer pipette) (2-20µl, 10-100µl, 100-1000µl)

Centrifuge.-

Shaking Water bath. -

Vortex.-

Spectrophotometer.-

slide4

15ml

50ml

Glassware

- Beakers (50ml, 80ml, 100ml, 500ml).

Volumetric flask (100ml, 250ml, 500ml, 1000ml).-

- Plastic and glass centrifuge tubes (15ml, 50ml).

- Measuring cylinders (50ml, 100ml, 500ml).

- Pasteur pipettes.

  • - Pipettes (1ml, 5ml, 10ml).

- Eppendorf tube.

- Tips with different size.

Racks.-

Quarts cuvettes. -

slide5

Reagent

Proteinase K (20mg/ml).-

Lysis buffer (2X). -

SDS 10%. -

Salt/ EDTA.-

- Chloroform: Isoamyl alcohol.

EDTA 0.5M. -

Ethanol 99-100%.-

10mM Tris, 1mM EDTA.-

1M Tris PH= 7.6-

- TE buffer 10:1

Phenol.-

slide6

Calculations

Number of moles (mole)

Weight (Gram)

Number of moles

Molecular weight (gram/mol)

Molarity

Volume (Liters)

Concentration X Volume = Concentration1 X Volume C X V = C1 X V1

calculations
Calculations

0.1 ml = 100µl

1 ml = 1000µl

100 ml = 10000µl

slide8

Extraction and purification of DNA

- The DNA was extracted manually from blood sample.

- This method uses SDS- proteinase K method which dissolve the the sample and digest the protein component without affecting the DNA.

slide9

Extraction and purification of DNA Procedure

Add 5ml of blood + 45ml of Lysis buffer(2X) to 50ml capped centrifuge tube.

Mix the samples using the Vortex for 10min.

Put the tubes in the Centrifuge for 10min at 3000rpm.

There will be 3 layers

slide10

Extraction and purification of DNA Procedure

Extraction and purification of DNA Procedure

Discard the supernatant.

Add 3ml of EDTA salt buffer + 0.3 ml of 10%SDS + 0.1ml of proteinase K to the pellet.

Incubate all the tubes over night at 37ºC in shaking water bath.

slide11

Extraction and purification of DNA Procedure

Extraction and purification of DNA Procedure

Add 3ml of liquid phenol to each tube.

Mix the samples using the Vortex for 10min.

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

There will be tow layers.

slide12

Extraction and purification of DNA Procedure

Extraction and purification of DNA Procedure

Add 3ml of chloroform:Isoamyl alcohol to the upper aqueous phase.

Put the tubes in the centrifuge for 5 min at 2000rpm.

Tow layers will appear.

Add 6ml of ethanol to each tube to precipitate the DNA.

slide13

Extraction and purification of DNA Procedure

Extraction and purification of DNA Procedure

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

Add 0.5 ml of 10mM EDTA buffer in 2 ml eppendorf tube to redissolve the DNA over night.

slide14

Measure of DNA concentration

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.

any question

Any Question?

Thank You