Strawberry DNA Plant Genomics

Genomics – The study of DNA Plant chromosomal DNA Chromosome number Plant genes Plant reproduction Plant gene expression – the regulation of genes

Why Plant Genomics Agricultural applications – Production of healthier crops- more nutritious( Genetic engineering of crop plants Production of crops with disease resistance Pharmacology - What novel genes do plants have to apply to human pharmacological research? Many contain anti- cancer compounds Bioremediation – Plants removing pollutants from the environment

Genomic DNA 30,000 genes Satellite DNA – around the centromeres Telomeric DNA – repetitive copies of TTAGGG VNTR – variable number of tandem repeats- variable per species Retrotransposons – remains of ancient retroviruses – are capable of replicating and making enzymes capable of jumping our of one position and finding a new position in DNA

Genomic DNA Sines- short interspersed elements – 500 bp but are not translated Lines – long interspersed elements – up to 7000 bases some are transcribed and translated Transposons- move around in the DNA ALUs – a special type of DNA – 300 bp – accounts for 11 % of the human genome

Contradictions to the Central Dogma Retroviruses – Other RNA viruses Transposons Other elements in DNA - Alus Prions ( proteins – Mad Cow) Genes for t- RNA Genes for Ribosomal RNAs Spliceosomes and catalytic RNA’s Enhancers and repressors Promoters

Materials Clean blue tube Sharpie marker Eppendorf holder( Microfuge tubes) Loading dye ( green and yellow tube) Tracking dye – white tube TD Marker – white tube – M Strawberry DNA- sample from extraction

Extraction of DNA I Homogenize strawberries Filter the stawberry homogenate Place extract in Corning Tube Add lysis mixture – detergent containing lauryl sulfate and salt, NaCl Add papain mixture to denature proteins( DNAses) Mix by rocking and rolling

Extraction of DNA II Heat at 55 o C. This speeds up degradation of proteins( 2 min) Place in ice until cold Add ice cold ethanol. Drip slowly down the side of the Corning tube making sure that the alcohol forms a layer on top of the juice. This should form an interface between the two layers.

DNA Precipitate III The DNA should precipitate and form a mass of slender,sticky strands Remove the DNA from the Corning tube, being careful not to disturb the interface The DNA should be placed in a microcentrifuge tube, Centrifuge for 2 minutes

Pellet and Supernatant Spin the tube with the DNA It forms a pellet on the side of the microcentrifuge tube Pour off the supernatant which is alcohol The DNA needs to be resolubilized in Tris buffer Mix the DNA from the pellet with Tris

Tris and DNA The DNA must be in solution for electrophoresis. Mix before using in the gel Remove 25 ul of DNA and place in a microcentrifuge tube. Add 3 ul of loading dye Vortex to mix

Genomic DNA – Gel Lanes Label on your index card 1-Tracking dye 2- Marker – Lambda phage - HindII Strawberry DNA samples( genomic DNA) Name on Ziplock Snack Bag for Gel

Loading gels Run DNA from black to red From the cathode to the anode Remember DNA has a negative charge

Reminders about loading gel Aspirate to first stop point Deliver by dispensing into the well to the second stop point Fill the well – being careful not to overfill Run the gel on a voltage of 80 volts

Staining Stain with Methylene blue. Fast Blast Stain. Gel should stain for at least one hour Destain with Distilled water Place gel in ziplock bag with water. When gel is sufficeintly destained - the stained gel can be placed in a ziplock for storage in the refrigerator

Gel Observation Observe gel on light box Measure the distance that each band in the marker( standard) migrates Measure any bands oar streaks of DNA you observe. Use the DNA graphing program to make a semi-log graph to estimate the sizes of your DNA

DNA gel