What is DNA. DNA DNA is the genetic material. Is Is a double helix. Made Made up of subunits called nucleotides Nucleotide

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1 What is DNA DNA DNA is the genetic material. Is Is a double helix. Made Made up of subunits called nucleotides Nucleotide made of: 1. hosphate group 2. 5-carbon sugar 3. Nitrogenous base 2 O 4' T 1' to 2' O O 2 O 4' 1' 2' O O 2 O G 4' 1' 2' O 1

2 2 O T O 4' 1' 2' to 2' 2 O 1' O 2 4' 2' 2 1' 4' O A to G 4' 1' 2' 2' 2 O G 1' O 2 4' O O 4' 1' 2' O DNA replication start with primer 2

3 O G RNA primer O G O A U U oops! G A A A G A G A DNA polymerase ( -> polymerase) ( -> exonuclease) O O T O O O O O O O O T Direction of replication 5` 3` DNA has proofreading Where is DNA in the cell? DNA extraction Most DNA extraction protocols consist of 5 parts: 1 A technique to lyses the cells gently and solubilize the DNA 2 DNA degrading enzymes must be deactivated 3 Enzymatic or chemical methods to remove proteins, lipids, RNA, or macromolecules. 4 DNA must be precipitated in alcohol solution 5 Washing : DNA must be precipitated in alcohol solution, then washed, dried, and resuspended in buffer or sterile water. 3

4 Overview of DNA Extraction Break down the cell wall and membranes entrifuge to separate the solids from the dissolved DNA recipitate the DNA using isopropanol Dissolve DNA Wash the DNA pellet with Ethanol and dry the pellet entrifuge to separate the DNA from the dissolved salts and sugars ell Lyses TA (or SDS) Remove lipids Denature proteins roteinase K (65 ) Digest protein Inactivate DNAse Remove istones hloroform/isoamyl alcohol (phenol) Remove proteins from DNA and RNA DNA precipitation and washing recipitation: 2 3 volumes cold ethanol Washing: ethanol 70 % Elution : TE buffer Water DNA spooling with glass 4

5 DNA extraction from different samples SAMLE REARATION Blood: 500µl in 1ml water, centrifuge at 5000rpm/2min and the pellet resuspended in 200µl of TE buffer. Sera: stored at -20. ells/bacteria : ells collected by centrifugation 7500rpm/10 min and resuspended in 200µl of TE buffer. Tissues: 30mg of mammalian tissue or mg of plant tissue grounded in liquid nitrogen with mortar and pestle. The powder resuspended in 200µl of TE buffer. Fermentas method Mix 200µl of sample with 400µl of lysis solution and incubate at 65 for 5min. add 600µl of chloroform, mix gently and centrifuge 10,000rpm/2min. Transfer the upper aqueous phase to a new tube. add 800µl of precipitation solution, mix gently/2min centrifuge at 10,000rpm for 2min. Remove supernatant completely and dissolve DNA pellet in 100µl of 1.2M Nal solution by gentle vortexing. Add 300µl of cold ethanol, let the DNA precipitate (10min at - 20 ) and spin down (10,000rpm, 3-3 4min). our off the ethanol Wash the pellet once with 70% cold ethanol and dissolve DNA in 100µl of sterile deionized water by gentle vortexing. 5

6 DNA extraction for student training: Nucleic Acid Storage <4 Months 1 3 Years <7 Years >7 Years 5 to Recommended in water or ethanol Recommended for long- term storage in ethanol Evaluation of Nucleic Acids (1) spectrophotometrically quantity quality 6

7 Example Experimental sample volume of 100 ul, diluted 100-fold (1 ul DNA + 99 ul water) Blank sample volume of 100 ul, diluted 100-fold (1 ul TE buffer + 99 ul water) OD260 = OD280 = DNA quality and concentration oncentration: (0.185)(50 ug/ml)( ) ) = 925 ug/ml = (0.925 mg/ml ml) Quality : OD260/OD280 OD280 = 0.177/ = 1.87 ( good purity bet ) Analyzing DNA samples With gel electrophoresis Analyzing DNA samples With gel electrophoresis Analysis of samples: (A): This sample is fine (B): This sample is fine () : This sample is fine (D) : This sample is fine (E): This sample has severe degradation, but can work for R Ladder A B D E 7

8 Analyzing DNA samples With gel electrophoresis The DNA below is a very high molecular weight, clear, thick band. This DNA was extracted in a research lab under optimal conditions This DNA is ideal for GE and sequencing. No RNA contamination No DNA degradation 1 kbp and 100 bp ladders Genomic DNA of 5 species of cereals DNA Quality from Agarose Gel Electrophoresis uman Whole Blood DNA Lambda DNA marker Lambda DNA cut with ind III marker Whole blood genomic DNA Electrophoresis of DNA Gel DNA Size 8

9 DNA Size from Agarose Gel Electrophoresis: ompares unknown DNA to known size standards Lambda DNA cut 1 kb ladder Lambda DNA with ind III 12,218 bp 100 bp ladder 23,130 bp 6,018 bp 1,500 bp 9,416 bp 6,557 bp 4,361 bp 3,054 bp 2,036 bp 1,636 bp 1,000 bp 600 bp 300 bp 48,500 bp 1,018 bp (48.5 kb) 100 bp 2,322 bp 2,027 bp 517 bp Troubleshooting Nucleic Acid reparation Methods roblem: No or low nucleic acid yield. Make sure that sample time was allowed for resuspension or rehydration of sample. Repeat isolation from any remaining original sample (adjust procedure for possible low cell number or poorly handled starting material). oncentrate dilute nucleic acid using ethanol precipitation. Troubleshooting Nucleic Acid reparation Methods roblem: oor nucleic acid quality If sample is degraded,, repeat isolation from remaining original sample, if possible. If sample is contaminated with proteins or other substances, clean it up by re- isolating (improvement depends on the extraction procedure used). 9

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