012814 replication and dna extraction hon bio 13-14
TRANSCRIPT
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The Significance of Watson & Crick
Provided an explanation
for how all the parts
of DNA could be
arranged in a mannerthat would allow the
molecule to replicate
itself
1stPart of Central
Dogma
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The Complimentary Base Pairing Of The Nitrogenous
Bases Is Due To The Number of Hydrogen Bonds
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During prophase, the supercoils of DNA may be
further condensed into chromosomes
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How Does DNA Copy Itself?
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REPLICATION OF DNA
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REPLICATION DNA molecule uncoils from tightly condensed state,
assisted by DNA Helicase. This unzipping occurs at the
replication fork. Hydrogen bondsbetween nitrogenous bases break
New exposed half-rungs of ladder-like structures are
called TEMPLATES
Free DNA nucleotides in the nucleus are brought into
place by DNA POLYMERASE
Due to complimentary base pairing, only free Adenines canpair with Thymines in the template, only free Guanines can
pair with Cytosines in the template, etc.
DNA Polymerase also proofreads the strands for mis-
paired nucleotides.
The DNA fork is recoiled
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Why Is It Essential That The Hydrogen Bonds Between
Nitrogenous Bases Be Weak?
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Replication Occurs In Two Directions
Simultaneously
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Lab: Extraction of DNA
Purpose: To ascertain and appreciate several
properties of DNA based upon the processes
required to extract DNA from the nucleus of a
eukaryotic cell.
Methodology: Proteolytic enzyme extraction
and separation using non-polar solvent
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Wheat Germ
Source of Nucleic Acids
Wheat germ is derived
from the part of the
wheat seed that isundergoing high mitotic
activity
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Lauryl Sulfate
Active ingredient in
dishsoaps
Why necessary to
disrupt the plasma andnuclear membranes?
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Heat
Why is it important to
have heat?
Why is it important not
to exceed 50C?
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Papain & Bromelin
Proteolytic enzymesfound in meattenderizer.
Derived from bromeliadplants (i.e. pineapple)that use these enzymesto ward off parasites
Problem: Meattenderizer also containsnucleases
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Sodium Hydrogen Carbonate
NaHCO3
Disassociated sodium ionsbind to negative aspectsof DNA polymer,
protecting it fromnucleases in meattenderizer
Can produce carbonicacid when it
disassociates. Why mightthis be useful in terms ofpapain?
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Lab Protocol
Add 45mL tap water to a 100 mL test tube and
place in a hot water bath. Heat until the
temperature of the water INSIDE THE TEST
TUBE is 55C.
DO NOT, AT ANY POINT, EXCEED 60C!
Add 2g wheat germ and 3mL of lauryl sulfate
(detergent) to the 45mL of water in the test
tube. Stir and incubate for 5 minutes
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Lab Protocol 2
After the first incubation, add 2g of Papain(meat tenderizer) and 5mL 1M NaHCO3. Stirand incubate at 55C for 15 minutes.
NEW STEP: Filter the liquid portion of themixture through a coffee filter into a 100 mLbeaker.
Cool the beaker to 25 C in an ice bath When ready, instructor will layer in 20mL iced
ethanol. Let stand 3-5 minutes and observe.