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What is the proper use of a micropipette? NVR: Adjust pipette below minimum 0.5 mnrL. or above maximum 10.0 mnrL.2: Use pipette w/o tip in place. It can ruin precission piston that measures the volume.3: Lay down pipette with tip on. Fluid can run back into the piston.
What is the relationship between the number of RECOGNITION SITES & the number of DNA fragments produced by different restriction enzymes? Recognition site is always one lelss number
What is the procedure used to separate DNA fragments? Agarose Gel Electrophoresis
To prepare DNA for profiling what is needed? 1. Specific Restricition Enzymes called: Endonucleases
DNA Double stranded genetic molecule that consists of many monomers called NEUCLEOTIDES.
DNA is made of double stranded genetic molecules that consists of many Neucleotides monomers. Hence, DNA is? A POLYNEUCLEOTIDE
How do the two strands of DNA connect to one another? By Hydrogen Bonds between the nitrogenous bases of each strand.
Is the BASE PAIR TOTAL the same every time? No it varies from species, to species.
How does the BASE PAIR TOTAL varies? By having fewer but measurable differences within the species.
Is it possible for two organisms to have the same BASE PAIR SEQUENCE? No. Unless they are twins or clones.
How can BASE PAIR SEQUENCE be used to identify genetic similarities? By using DNA from two sources using a procedure known as DNA PROOFILING
What is DNA PROFILING? The procedure used to identify genetic similarities by using DNA from two sources,
What does using RESTRICTION ENZYMES (EDONUCLEASES) in DNA profiling do? Cuts both strands of DNA at a specific sites known as recognition sites.
RECOGNITION SITES Specific sites where both strands of DNA are cut.
What is the Restriction Map? shows exact locations of recognition sites for a particular resriction enzyme on a DNA source.
What is the theory behind agarose gel electrophoresis? (separation based on charge) Electrophoresis "carry with an electric current"Digested DNA is loaded into an agarose gel and an electric current is passed across the gel.DNA is a negative charged molecule that will migrate through the gel towards the positive pole of the chamber.Will
What is Migration Distance? smaller DNA fragment=separates farthest due to less friction
ELECTROPHORESIS means "to carry with an electric current"
Why is DNA negatively charged? Because of the presence of LARGE numbers of PHOSPHATE GROUPS in its backbone.
ELECTROPHORESIS CHAMBER Chamber containing Agorose Gel
How is the DNA Fragments arranged? By Molecular Size
What happens to the fragments during the procedure ? smallest fragments will migrate farther in the gel. Larger fragments will lag behind.
What makes these fragments visible? staining.
What presents the DNA PROFILE? The pattern of stained DNA "bands".
DNA RESTRICTION MAP indicates the exact locations of the recognition sites for a particular restriction enzyme on a DNA source.
What was the BUFFER SOLUTION used in the DNA Lab? EBT
What did the BUFFER do? it fluoresces under UV light so you can see the location of the DNA
What are two functions of the loading dye? a.To make DNA visible b.To allow the sample of DNA to sink into the wells.
How does ethidium bromide stain DNA? When excited by UV light, it resonates and gives off fluorescent light. As a result of its planar ring structure which slips between the bases of a strand of nucleic acid.
why should we minimize exposure to ethidium bromide ? Because : EtBr slips into DNA it can be construed as a base by DNA or RNA polymerase. This will cause frameshift and nonsense mutations by adding bases to replicated DNA or newly synthesized RNA.
why should we minimize exposure to ethidium bromide ? Because: Ethidium bromide is mutagenic, causes damage to DNA. Exposure to it (skin contact or inhalation) should be avoided as it will damage the DNA in the cells it comes into contact with and hence have a negative effect on the cell.
What will occur to to the gel box if water is used instead of the TBE BUFFER? It will not get a current
What will occur to to the gel box if the electrolodes are reversed? Carries sample off gel
How can you account for differences in separation and intensity ? properly forming your well=no bubbles properly micropipetting contents into the wells don't puncture the wells, or under/overload the wells; set electrophoresis to proper time + voltage to create the ideal amount of separation between DNA fragments.
What affect will be observed in the stained bands of DNA in an agorase gel if casting tray is removed or jarred while agorose still solidifying? Creates currents thick or thin
What affect will be observed in the stained bands of DNA in an agorase gel if is ran at a very high voltage? denature DNA molecule+ melt gel
What affect will be observed in the stained bands of DNA in an agorase gel if a larger air bubble or clump is allowed to set in agorose? Deformed and smears
What affect will be observed in the stained bands of DNA in an agorase gell if too much DNA is loaded in a lane? Short runs
DOUBLETS DNA Fragments that won't resolve in the gel. Can be recognized because they are brighter than the regular fragments.
What is it's size?
What percentage was the Agorase Gel that you ran?
What was used to stain the DNA bands? ETB
What was the purpose of the control? to show organic growth
Is it necessary to use a fresh tip when delivering the same reagent? yes because cotamination might happen.
Why was water added to the control tube?
How does TBE stain DNA? sliping between base pairs in DNA + stays there
ELECTRODES two conections of the electrophoresis box machine
Voltage 75 V.
electrophoresis 1hr and 15 minutes
Illumination of Electrophoresis Product 1. Power supply off/ remove lid from gel chamber 3. Wearing gloves/Pick up gel tray +gently slide the gel into plastic container. 4.Let gel to cool off 5mins 5.Bring Agorose Gel to UV Light BOX.
Illumination of Electrophoresis Product (2 part) 6 Power off+raise lid of light box+spatula 2 place gel in center of light field 7.Close lid+Tun on light box. (lights off in the classroom) 8. View pattern of DNA file (DNA Profile) 9. Draw the approximate location of each DNA bands
Illumination of Electrophoresis Product (2 part) 10. finished:Turn off light box+Spatula discard gel into disposal
cathode black
anode red
Negative pole on the chamber black dot
What are the names of the monomers that make up DNA? Nucleotides Adenine, Thymine Guanine, Cytosine
Function of compromise restriction buffer? TBE buffer carries current through entire system and maintains pH
Why is restriction cell important to bacteria? found in bacteria/ participate's in cell defense/ “restrict” foreign DNA that enters the cell, by destroying it.
What is the source of the DNA used in this experiment? (Lab 9) lambda bacteriophage
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