Lab Practical 1 BIO208 spring 2009The Laboratory NotebookBioinformatics - Review the worksheet from week 1BioinformaticsQuestions: (also see pre-lab)How is lysis accomplished?The p20 is used. What is this volume in microliters (l)? Lab Practical 1 BIO208 spring 2009The Laboratory Notebook Bioinformatics - Review the worksheet from week 1BioinformaticsNCBIBLASTGenBankFASTA formatIdentity and function of each protein in the folklore recipeDNA Isolation Review the pre-labTerms Reagents (what does each do?) EquipmentLysisFiltrationPrecipitationSolubilize SpectrophotometryHigh molecular weight DNADenaturation of endonuclease enzymesPhospholipid bilayerCentrifugationSupernatantPelletWater/Ethanol interfaceTemperatures (0oC, 22 oC, 37 oC,100 oC)Detergent (SDS)Ethanol (ice-cold)WaterNaClPlant seedsWaterWater bathDigital balanceTriple beam balanceCheeseclothUV SpectrophotometerCuvetteCentrifugeModel of DNAQuestions: (also see pre-lab)How is lysis accomplished?How is precipitation of DNA accomplished? Describe its appearanceHow are cellular lipids and proteins solubilized?How is high molecular weight DNA stored?How are endonucleases, enzymes that destroy DNA, denatured?What is in the supernatant? The pellet?How much ice-cold ethanol would you add to precipitate a DNA sample of 20 ml?How is the concentration of DNA calculated using the 50ug/ml = OD 1.0 formula?What material absorbs a UV wavelength of 260? 280?What 260/280 ratio indicated pure DNA?If 100 microliters of DNA is added to 400 ul of sterile water and the OD reading is 0.52, then what is the actual concentration of DNA (take into account the dilution factor)?If a yield of 655 ug of DNA is obtained and 20 grams of strawberries were used initially, what is the yield of DNA per gram of tissue?What is the Beer-Lambert law of spectrophotometry?Does DNA dissolve in water or in ethanol? (pick one)What is room temperature, body temperature, boiling, a refrigerator, and freezing in degree Celsius?How are tubes balanced in the centrifuge?At what speed (rpm) is precipitated DNA centrifuged?DNA Fingerprinting Review the pre-labTerms Reagents (role of each) EquipmentPV-92, Alu element941 bp and 641bp PCR productsDimorphic, monomorphicHomozygous +/+Heterozygous +/-Homozygous -/-Taq polymerasePrimersDenaturation of DNAAnnealing of primersExtension of DNA Cheek epithelial cellsIsotonic LysisChelexSalinePCR mix – primers, nucleotides, MgCl2, buffer, Taq polymeraseTemplate DNAAgarose1X TBE bufferLoading dyeDNA size markers (EX load)Ethidium bromide ThermocyclerWater bathMicrocentrifuge (microfuge)p20, p200, p1000 pipettemenBlue and yellow tipsPower supplyElectrophoresis equipment (tanks, combs, tape, microwave)UV TransilluminatorMicrowaveDigital balanceQuestions (also see pre lab)Why is saline used to isolate cheek cells?What is the appearance of the cheek cell pellet? What is in the supernatant?What is the role of Chelex in the template DNA preparation? Why remove it? What is in the supernatant after the Chelex centrifugation?The p20 is used. What is this volume in microliters (l)?  The p200 is used. What is this volume in l?--> What occurs during the 94 oC step in the PCR?What occurs during the 60 oC step in the PCR?What occurs during the 72 oC step in the PCR?What is a primer?Why would a cycling of 94 oC -> 72 oC -> 60 oC not work in the PCR?0.5 ug of DNA is needed to visualize on a gel. How many PCR cycles are needed to obtain 0.5 micrograms DNA from an initial amount of 2 nanograms (ng) (there are 1000ng in a microgram).Where was taq polymerase isolated from and what does this enzyme do?Why is the cheek cell prep heated at 56 oC and then boiled?What is the role of loading dye in electrophoresis? Why is glycerol in the loading dye?What is a size marker?What is ethidium bromide used for and how does it work?What is a typical voltage at which to run a gel? What is a typical percentage of agarose in a gel?How do you make a 0.8% gel in 100 ml of 1X TBE (wgt/volume)?Which individuals could be the father of baby #1? baby #2?What is the genotype of each individual (all 6 of them) with respect to the Alu element, PV 92? ?Size marker baby #1 baby #2 mother Simon Jose Steve __1000bp ____ ____ ____ ____ ____ __700 bp ____ ____ ____ ____ __400bp