Minutes 20100927 Moffatt BIOL 208

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Quiz Questions / Tutorial Questions

Proteins that bind the most strongly in chromotography elute first (74% correct).
Questions were done well overall.
Complaint: tutorial not helping with tutorial quiz
Uncertain if it's just some students?

OD260 calculations
Spectroscopy
Notice the numeracy required within the quiz questions
Sequence of the primers
Synthesizing a strand "this is the sequence we are making" (not the template for the enzyme)
Precipitation of DNA -- salts, aggregates, etc.,
Rinsing with ethanol
P60 of notes -- figure of steps: go over the steps again.
Pellet on the inside -- does it form into a haze instead? White pellet bad -- salt, protein; translucent => mostly DNA
Wash: "cold 70% and spin again, remove supernatant and dry."
Centrifugation is a way to recover things -- keep either supernatant or pellet depending on what you want
Plasmid prep
What is EDTA? Keeps DNA intact
"Chelate", "Sequester", "Bind"
Mg⁺⁺ is an enzyme cofactor -- chelating (sequestering) Mg⁺⁺ keeps DNA safe
Buffer (tris, etc.,) does not include DDT, Glycerol etc. -- Mg⁺⁺ is part of the buffer
Autoclaving -- sterilization
Trying to recover something -- RNAse free DNA / DNAse free RNA
Boil RNAse stock to deactivate DNAse (RNAse is thermally stable; can even be autoclaved -- high temperature, high pressure)
RNAse is deactivated chemically -- expensive item that you must buy
Phenol is used to destroy proteins -- you cannot recover proteins from phenol (dead, aggregated, denatured)

See p.62 -- Header 4
(10 µg is the solution for p.63)
Go over an example calculation -- similar to molar calculation
See lecture slides
Poll for the most difficult questions / terminology and cover those?
- Dr. Moffatt is working on this item