Presentation on theme: "Restriction Enzyme Digest"— Presentation transcript:
1 Restriction Enzyme Digest Digesting E. huxleyi genomic DNA
2 Single or Multicopy gene in Emiliania huxleyi? MetacaspaseSingle or Multicopy gene in Emiliania huxleyi?
3 Research Plan Isolate Genomic DNA Southern Blot Analysis Digest Genomic DNA w/ Various Restriction EnzymesAgarose Gel Electrophoresis and Southern TransferMake Non-Radioactive Metacaspase ProbeHyribidize Probe to Southern BlotWashes and Chemiluminescent DetectionData Analysis
4 BRAVO!!!YOU DID IT!!!You Isolated GenomicDNA from E. huxleyi!!!
5 Today’s Laboratory Objectives Determine the concentration, purity, and integrity of the E. huxleyi genomic DNADigest E. huxleyi genomic DNA
6 Theoretical Basis of UV Spectrophotometry A UV spectophotometer measures the amount of light particular molecules absorb (Proteins at A280; Nucleic Acids at A260)Lambert-Beer law describes the relationship between absorptivity coefficient and concentration and is given by the following equation:A=εbcWhere: b= light path lengthc=concentration of substanceε=extinction coefficientFor DNA the extinction coefficient, ε= 50 ug/ml
7 Theoretical Basis of UV Spectrophotometry To Quantify your DNA sample:A260 x Dilution Factor x 50 ug/ml= concentration of nucleic acids in a sample using a 1 cm pathlengthTo estimate the purity of your sample:A260/A280= ratio of nucleic acids/proteinA260/A280= is optimal for DNA
8 Theoretical Basis of Agarose Gel Electrophoresis Agarose is a polysaccharide from marine alage that is used in a matrix to separate DNA moleculesBecause DNA ia a (-) charged molecule when subjected to an electric current it will migrate towards a (+) pole
10 Sizing a Piece of DNA The distance the DNA migrates is dependent upon the size of the DNA moleculethe secondary structure of the DNAthe degree of crosslinking in the gel matrixSize of DNA molecule can be determined by using standards of known molecular weighta standard curve is made by plotting the molecular weights of thestandards and the distance each fragment has migrated from the measuring the distance the unknown fragment migrated from thewellsubstituting the distance the unknown migrated into the equation ofthe line of best fit, and solving for Y (the molecular wt)
11 Assessing the Integrity of DNA High Quality Genomic DNA>95% DNA will be of high molecular weight, migrating as intact band near the top of the gelVery little evidence of smaller fragments indicated by a smear of many different sized DNA fragments
12 Restriction Enzymescalled "restriction enzymes“ because restrict host range for certainbacteriophagebacterial" immune system": destroy any "non-self" DNAmethylase recognizes same sequence in host DNA and protects it by methylating it; restriction enzyme destroys unprotected = non-self DNA (restriction/modification systems)
13 Restriction EnzymesHundreds of restriction enzymes have been identified.Most recognize and cut palindromic sequencesMany leave staggered (sticky) endsby choosing correct enzymes can cut DNA very preciselyImportant for molecular biologists because restriction enzymes create unpaired "sticky ends" which anneal with any complementary sequence
14 Some Commonly Used Restriction Enzymes Eco RI 5'-G | AATTCEco RV 5'-GAT | ATCHin D III 5'-A | AGCTTSac I 5'-GAGCT | CSma I 5'-CCC | GGGXma I 5'-C | CCGGGBam HI I 5'-G | GATCCPst I I 5'-CTGCA | G
15 Theoretical Basis Using Restriction Enzymes The activity of restriction enzymes is dependent upon precise environmental condtions:PHTemperatureSalt ConcentrationIonsAn Enzymatic Unit (u) is defined as the amount of enzyme required to digest 1 ug of DNA under optimal conditions:3-5 u/ug of genomic DNA1 u/ug of plasmid DNAStocks typically at 10 u/ul
16 Next WeekSeparate our restriction fragments using agarose gel electrophoresisSouthern Transfer- transfer denatured DNA from agarose gel to a membrane on which it can be analyzed using a labelled complementary DNA probe to metacaspase