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BIOL239 Study Guide - Quiz Guide: Replica Plating, Molecular Cloning, Southern BlotPremium

7 pages94 viewsWinter 2017

Department
Biology
Course Code
BIOL239
Professor
Christine Dupont
Study Guide
Quiz

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Genetic Analysis
Things to recall with a library?
oLibraries are used to track a specific fragment, but in order to determine which
cell has it use probes to bind to complimentary gene areas (probes are single
stranded)
oThey isolate fragments that wouldn’t normally be isolated as they are in a smear)
Describe probes in more detail
oShort single-stranded stretches of DNA of known composition, form 25 to several
thousand nucleotides in length
o1) To visualize them, the probes are labeled, usually with 32P or fluorescent dyes
Used in the identification of clones that contain complementary DNA sequences.
From 25 to several thousand nucleotides
in length
oSeveral ways to make labeled probes Can be
chemically synthesized, and are
termed
oligonucleotides
(usually 25 to 100
nucleotides in length).
Fluorescent dyes can be added as part of the
synthesis procedure.
oMust know the DNA sequence of the probe
o2) Can label a previously cloned fragment
Removal from the vector by restriction endonuclease
digestion, followed by chemical
labeling
usually 100 - 500 base pairs in
length
Don't need to know the sequence of this probe
Error! Filename not specified.
Describe screening through hybridization
oIsolating gene
Replica plating allows to find exact l
ocation on master plate
oNylon adheres to DNA, all junk can be washed
away
oNaOH used to break hydrogen bonds
and make DNA single stranded
oThis break allows probe to bind to
complementary gene
oLook where different on replica plate, see where is
on master plate to find gene of interest
Get mRNA for gene of interest --> Make it cDNA (single stranded) --> Get colony
library which has normal DNA and your inserted vector from before --> Replica to nylon
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--> Kill cells to expose DNA --> NaOH to make single --> add cDNA probes --> Wash
away non annealled stuff --> X ray to see where colony with vector of interest is located
--> Refer to master plate
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Evaluate screening through hybridization
oThe region of complementarity between probe and target sequence
must be long enough to allow sufficient hydrogen bonds to provide a
cohesive force
oIn general, two single DNA strands that are longer than 50-100
bp will hybridize so long as the extent of their complementarity
is more than 80%
needs to fit both parameters to work
Error! Filename not specified.
Describe Southern Blot Analysis
oUsed to show the presence of a gene within a larger fragment of DNA Involves
the transfer of DNA fragments from an agarose gel to nitrocellulose filter paper
oThe relative position of the DNA fragments is preserved in the transfer
Hybridization between a specific probe and DNA on the filter can be carried out
oNot isolating gene, just want to see if have it
oNylon membrane makes stick in place (not diffuse once electrogradient is
removed) render single stranded w/ NaOH
oCapillary action of buffer below goes up imprinting DNA to nylon membrane.
Paper towels for capillary action
oShows if an individual has a gene, if so how frequent is it?
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