EXTRACTING DNA FROM WHOLE BLOOD

EXTRACTING DNA FROM WHOLE BLOOD4BIO2 - Group 1 November 4, 2014INTRODUCTIONBLOODabodily fluidinanimalsthat delivers necessary substances such asnutrientsand oxygento thecellsand transportsmetabolic wasteproducts away from those same cellscomposed ofblood cellssuspended in blood plasmaWhole blood plasma and cells togetherDNA EXTRACTIONprocess of isolating of DNA from sample using a combination of physical and chemical methods.Follows the process ofCell lysisDNA purificationDNA resuspensionAGECELL LYSISto expose the DNA within the cellremoval of membrane lipidscommonly achieved by chemical and physical methods-blending, grinding orsonicatingthe sampleCell lysis bufferDetergentsSurfactants

PROTEIN PRECIPITATIONDegradation of proteins associated with DNAAchieved by the addition of a proteaseProtease K - digest protein and remove contamination from preparations ofnucleic acidrapidly inactivatesnucleasesthat might otherwise degrade the DNA or RNA during purificationaided by the addition of a salt such as ammonium or sodium acetatePHENOL-CHLOROFORM EXTRACTIONphenoldenaturesproteins in the sampledenaturated proteins stay in organic phase aqueous phase containingnucleic acidis mixed with thechloroform

DNA RESUSPENSIONDNA is insoluble in the alcohol and will come out of solutionalcohol serves as a wash to remove the salt previously addedDNA can be re-suspended in a buffer such as Tris or TETE - solubilize DNA or RNA, while protecting it from degradationAGAROSE GEL ELECTROPHORESISseparate a mixed population of DNA or proteins in a matrix ofagarosebiomolecules separated by applying anelectric fieldto move the charged molecules through anagarosematrixbiomolecules are separated by size in the agarose gel matrix MATERIALS & METHODOLOGYMATERIALSWhole BloodPhenol / chloroform / isoamyl alcohol (25:24:1)Chloroform / isoamyl-alcohol (24:1)Sodium acetate, (CH3COONa) 3MSodium dodecyl sulfate, (SDS) 20%Proteinase K (10mg/ml)Isopropanol (2-Propanol)Lysis bufferSE bufferTE buffer

MATERIALSPhenolEthanol, 70 %Agarose gel, 1%Ice

EQUIPMENTDryblock heaterRefrigerated centrifugeUv-vis spectrophotometerMicrocentrifuge tubesMicropipettors and tips

RESULTS AND DISCUSSION GUIDE QUESTIONS1.) What is the role of the following In DNA extraction?Ethanol wash to remove salt previously addedNaCl Degradation of proteins associated with DNASDS Cell lysis; detergentTE Buffer - solubilize DNA or RNA, while protecting it from degradationEDTA CELL LYSIS: chelates cations that may bind to the negatively-charged DNA

2.) Give the complete CHEMICAL names of the following: SDS Sodium dodecyl sulfateTris Buffer tris (hydroxymethyl)aminomethaneEDTA Ethylenediaminetetraacetic acid3.) 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?