EXTRACTIN

G DNA

FROM WHOLE

BLOOD

4 B I O2 -

G R O U P 1 – N

O V E M B E R 4, 2

0 1 4

INTRODUCTIONBLOODa bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells

composed of blood cells suspended in blood plasma

Whole blood – plasma and cells together

DNA EXTRACTIONprocess of isolating of DNA from sample using a combination of physical and chemical methods.

Follows the process ofCell lysisDNA purificationDNA resuspensionAGE

CELL LYSISto expose the DNA within the cellremoval of membrane lipidscommonly achieved by chemical and physical methods-blending, grinding or sonicating the sample

Cell lysis bufferDetergentsSurfactants

PROTEIN PRECIPITATIONDegradation of proteins associated with DNAAchieved by the addition of a protease

Protease K - digest protein and remove contamination from preparations of nucleic acid

rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification

aided by the addition of a salt such as ammonium or sodium acetate

PHENOL-CHLOROFORM EXTRACTIONphenol denatures proteins in the sampledenaturated proteins stay in organic phase aqueous phase containing nucleic acid is mixed with the chloroform

DNA RESUSPENSIONDNA is insoluble in the alcohol and will come out of solution

alcohol serves as a wash to remove the salt previously added

DNA can be re-suspended in a buffer such as Tris or TETE - solubilize DNA or RNA, while protecting it from degradation

AGAROSE GEL ELECTROPHORESISseparate a mixed population of DNA or proteins in a matrix of agarose

biomolecules separated by applying an electric field to move the charged molecules through an agarose matrix

biomolecules are separated by size in the agarose gel matrix

MATERIALS & METHODOLOGY

MATERIALS• Whole Blood• Phenol / chloroform / isoamyl alcohol (25:24:1)• Chloroform / isoamyl-alcohol (24:1)• Sodium acetate, (CH3COONa) 3M• Sodium dodecyl sulfate, (SDS) 20%• Proteinase K (10mg/ml)• Isopropanol (2-Propanol)• Lysis buffer• SE – buffer• TE – buffer

MATERIALS• Phenol• Ethanol, 70 %• Agarose gel, 1%• Ice

EQUIPMENT• Dryblock heater• Refrigerated centrifuge• Uv-vis spectrophotometer• Microcentrifuge tubes• Micropipettors and tips

Transfer Supernatant into a new tube

Transfer Again the supernatant into a

new tube

-Add 100µl Chloroform/ Isoamly alcohol/ Phenol (25:24:1)- Shake by hand for 10 minutes- Centrifuge at 3000 rpm for 5 mins at 10 ° C

-Add 100µl chloroform/ Isoamyl-alcohol (24:1) -Shake by hand for 10 minutes-Centrifuge at 3000 rpm for 5 mins at 10 ° C

Transfer the Supernatant in to a new tube

Wash the DNA in 70% Ethanol

-add 30 µl 3 M sodium acetate (pH 5.2) and 100 µl isopropanol-Shake gently until the DNA precipitated

Use a glass pipette and make a hook over the Bunsen burner and capture the DNA

Dissolve the DNA in 50-100 µl TE- buffer overnight at 4°C on a rotationg shaker

Measure the DNA concentration in a Spectrophotometer and run 200 ng on a 1 % Agarose gel

RESULTS AND DISCUSSION

GUIDE QUESTION

S

1.) WHAT IS THE ROLE OF THE FOLLOWING IN DNA EXTRACTION?

A. Ethanol – wash to remove salt previously addedB. NaCl – Degradation of proteins associated with

DNAC. SDS – Cell lysis; detergentD.TE Buffer - solubilize DNA or RNA, while

protecting it from degradationE. EDTA – CELL LYSIS: chelates cations that may

bind to the negatively-charged DNA

2.) GIVE THE COMPLETE CHEMICAL NAMES OF THE FOLLOWING:

A. SDS – Sodium dodecyl sulfateB. Tris Buffer – tris (hydroxymethyl)aminomethaneC. EDTA – Ethylenediaminetetraacetic acid

3.) WHY SHOULD THE EXTRACTED DNA BE IMMERSED IN TE BUFFER?

The purpose of TE Buffer is to solubilize DNA while protecting it from degredation.

4.) WHAT IS THE ABSORBANCE OF DSDNA AT 260NM? SSDNA?