Strawberry DNAStrawberry DNA

Plant GenomicsPlant Genomics

Genomics – The study of Genomics – The study of DNADNA

Plant chromosomal DNAPlant chromosomal DNA

Chromosome numberChromosome number

Plant genesPlant genes

Plant reproductionPlant reproduction

Plant gene expression – the Plant gene expression – the regulation of genesregulation of genes

Why Plant GenomicsWhy Plant Genomics

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

Genomic DNAGenomic DNA

30,000 genes30,000 genesSatellite DNA – around the Satellite DNA – around the centromerescentromeresTelomeric DNA – repetitive copies of Telomeric DNA – repetitive copies of

TTAGGGTTAGGGVNTR – variable number of tandem VNTR – variable number of tandem repeats- variable per speciesrepeats- variable per speciesRetrotransposons – remains of Retrotransposons – remains of ancient retroviruses – are capable of ancient retroviruses – are capable of replicating and making enzymes replicating and making enzymes capable of jumping our of one capable of jumping our of one position and finding a new position position and finding a new position in DNAin DNA

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

Contradictions to the Central DogmaContradictions to the Central Dogma

Retroviruses – Other RNA virusesRetroviruses – Other RNA virusesTransposonsTransposonsOther elements in DNA - AlusOther elements in DNA - AlusPrions ( proteins – Mad Cow)Prions ( proteins – Mad Cow)Genes for t- RNAGenes for t- RNAGenes for Ribosomal RNAsGenes for Ribosomal RNAsSpliceosomes and catalytic RNA’sSpliceosomes and catalytic RNA’sEnhancers and repressorsEnhancers and repressorsPromotersPromoters

MaterialsMaterialsClean blue tubeClean blue tubeSharpie markerSharpie markerEppendorf Eppendorf holder( Microfuge tubes)holder( Microfuge tubes)Loading dye ( green and Loading dye ( green and yellow tube)yellow tube)Tracking dye – white tube Tracking dye – white tube TDTDMarker – white tube – MMarker – white tube – MStrawberry DNA- sample Strawberry DNA- sample from extractionfrom extraction

Extraction of DNA IExtraction of DNA IHomogenize strawberriesHomogenize strawberriesFilter the stawberry Filter the stawberry homogenatehomogenatePlace extract in Corning Place extract in Corning TubeTubeAdd lysis mixture – Add lysis mixture – detergent containing lauryl detergent containing lauryl sulfate and salt, NaClsulfate and salt, NaClAdd papain mixture to Add papain mixture to denature denature proteins( DNAses)proteins( DNAses)Mix by rocking and rollingMix by rocking and rolling

Extraction of DNA IIExtraction of DNA IIHeat at 55Heat at 55ooC. This speeds C. This speeds up degradation of proteins( up degradation of proteins( 2 min)2 min)Place in ice until coldPlace in ice until coldAdd ice cold ethanol. Drip Add ice cold ethanol. Drip slowly down the side of the slowly down the side of the Corning tube making sure Corning tube making sure that the alcohol forms a that the alcohol forms a layer on top of the juice.layer on top of the juice.

This should form an This should form an interface between the two interface between the two

layerslayers..

DNA Precipitate IIIDNA Precipitate IIIThe DNA should precipitate The DNA should precipitate and form a mass of and form a mass of slender,sticky strandsslender,sticky strands

Remove the DNA from the Remove the DNA from the Corning tube, being careful Corning tube, being careful not to disturb the interfacenot to disturb the interface

The DNA should be placed The DNA should be placed in a microcentrifuge tube,in a microcentrifuge tube,

Centrifuge for 2 minutesCentrifuge for 2 minutes

Pellet and SupernatantPellet and Supernatant

Spin the tube with the DNASpin the tube with the DNA

It forms a pellet on the side It forms a pellet on the side of the microcentrifuge tubeof the microcentrifuge tube

Pour off the supernatant Pour off the supernatant which is alcoholwhich is alcohol

The DNA needs to be The DNA needs to be resolubilized in Tris bufferresolubilized in Tris buffer

Mix the DNA from the pellet Mix the DNA from the pellet with Triswith Tris

Tris and DNATris and DNA

The DNA must be in The DNA must be in solution for electrophoresis.solution for electrophoresis.

Mix before using in the gelMix before using in the gel

Remove 25 ul of DNA and Remove 25 ul of DNA and place in a microcentrifuge place in a microcentrifuge tube.tube.

Add 3 ul of loading dyeAdd 3 ul of loading dye

Vortex to mixVortex to mix

Genomic DNA – Gel LanesGenomic DNA – Gel LanesLabel on your index cardLabel on your index card

1-Tracking dye 1-Tracking dye

2- Marker – Lambda phage 2- Marker – Lambda phage - HindII- HindII

3 -8- Strawberry DNA 3 -8- Strawberry DNA samples( genomic DNA)samples( genomic DNA)

Name on Ziplock Snack Bag for Gel

Loading gelsLoading gels

Run DNA from Run DNA from black to redblack to red

From the From the cathode to cathode to the anodethe anode

Remember Remember DNA has a DNA has a negative negative chargecharge

Reminders about loading gelReminders about loading gel

Aspirate to first stop Aspirate to first stop pointpointDeliver by dispensing Deliver by dispensing into the well to the into the well to the second stop pointsecond stop pointFill the well – being Fill the well – being careful not to overfillcareful not to overfillRun the gel on a voltage Run the gel on a voltage of 80 voltsof 80 volts

StainingStainingStain with Methylene blue. Stain with Methylene blue. Fast Blast Stain. Gel should Fast Blast Stain. Gel should stain for at least one hourstain for at least one hourDestain with Distilled waterDestain with Distilled waterPlace gel in ziplock bag with Place gel in ziplock bag with water.water.When gel is sufficeintly When gel is sufficeintly destained - the stained gel destained - the stained gel can be placed in a ziplock for can be placed in a ziplock for storage in the refrigeratorstorage in the refrigerator

Gel ObservationGel Observation

Observe gel on light boxObserve gel on light box

Measure the distance that each band Measure the distance that each band in the marker( standard) migratesin the marker( standard) migrates

Measure any bands oar streaks of Measure any bands oar streaks of DNA you observe.DNA you observe.

Use the DNA graphing program to Use the DNA graphing program to make a semi-log graph to estimate make a semi-log graph to estimate the sizes of your DNAthe sizes of your DNA

DNA gelDNA gel