Lab Practical 1 BIO208 spring 2009
The Laboratory Notebook
Bioinformatics - Review the worksheet from week 1
Bioinformatics
NCBI
BLAST
GenBank
FASTA format
Identity and function of each protein in the folklore recipe
DNA Isolation Review the pre-lab
Terms Reagents (what does each do?) Equipment
LysisFiltration
Precipitation
Solubilize
Spectrophotometry
High molecular weight DNA
Denaturation of endonuclease enzymes
Phospholipid bilayer
Centrifugation
Supernatant
Pellet
Water/Ethanol interface
Temperatures (0oC, 22 oC, 37 oC, 100 oC) / Detergent (SDS)
Ethanol (ice-cold)
Water
NaCl
Plant seeds
Water / Water bath
Digital balance
Triple beam balance
Cheesecloth
Spectrophotometer
Cuvette
Centrifuge
Model of DNA
Questions: (also see pre-lab)
How is lysis accomplished?
How is precipitation of DNA accomplished? Describe its appearance
How 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-Lambertlawof 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 thepre-lab
TermsReagents (role of each)Equipment
PV-92, Alu element941 bp and 641bp PCR products
Dimorphic, monomorphic
Homozygous +/+
Heterozygous +/-
Homozygous -/-
Taq polymerase
Primers
Denaturation of DNA
Annealing of primers
Extension of DNA
Cheek epithelial cells
Isotonic
Lysis / Chelex
Saline
PCR mix – primers, nucleotides, MgCl2, buffer, Taq polymerase
Template DNA
Agarose
1X TBE buffer
Loading dye
DNA size markers (EX load)
Ethidium bromide / Thermocycler
Water bath
Microcentrifuge (microfuge)
p20, p200, p1000 pipettemen
Blue and yellow tips
Power supply
Electrophoresis equipment (tanks, combs, tape, microwave)
UV Transilluminator
Microwave
Digital balance
Questions (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 72oC step in the PCR?
What is a primer?
Why would a cycling of 94oC -> 72oC -> 60oC 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 1000ngin 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 individualscould bethe 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