Final Lab Review All you need to know for lab MC in final exam!

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16 Oct 2011
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Lab 1 DNA Isolation
1. Mash up the material into small pieces
provide a greater surface area on which the chemicals can work
help to break down cell walls
2. Use detergent and heat to denature cellular proteins and remove protein bound to the DNA
use an enzyme to begin breaking the protein down into smaller peptide fragments so the
protein is less likely to precipitate with the DNA
remove large clumps of material and other cellular debris with centrifugation
3. Add a high salt solution and alcohol to precipitate your DNA while leaving most of the protein
in suspension.
4. SB- is a Squishing Buffer that contains neither SDS nor Proteinase K
SDS – solubilizes the plasma membrane as well as the nuclear envelope
SDS is a strong protein denaturant, which unfolds proteins and will dissociate both
histone and nonhistone proteins form DNA
Proteinase K – cleaves peptide bonds, thereby digesting and removing protein
contaminants from nuclei acids
5. yield - size of the pellet, purity – colour of the pellet
Lab 2 Restriction Enzymes and Agarose Gel Electrophoresis
1. restriction enzymes – site-specific enzymes
act as a defence system offering protection against foreign DNA such as viruses and
bacteriophages
the majority of restriction enzymes recognize either four or six base pairs
restriction map
2. Agarose Gel Electrophoresis
electrophoresis is used to separate molecules via an electric current
DNA and RNA carry a negative charge (due to the sugar-phosphate backbone) and move
towards the positive electrode when placed in an electric field
rate of migration – size, shape and charge-to-mass ratio
most DNA molecules are the same shape and all have very similar charge-to-mass ratio
the smaller the fragment, the faster it can travel through the gel
linear DNA fragments migrate at rates inversely proportional to their base-pair length
Lab 3 Library, Controls, and Evidence
1. A review article
Title, Author(s), Abstract (or Summary), Discussion as the main part and a list of
references
2. A primary research paper
Title, Author(s), Abstract (or Summary), Introduction, Materials and Methods, Results,
Discussion, and References
3. Experimental controls
positive control – positive response
negative control – unless the conditions and components are correct there is no response
no treatment control
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