Minutes 20100927 Moffatt BIOL 208
From SnOwy - Ed's Wiki Notebook
Contents |
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?
Chapter
- Cloning, Vector
- Genomic, PCR
- Purification, Quantification
- Phoresis, Standard Curves
Quiz Topics / Tutorial Content
- 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)
Tutorial / Quiz / Calculations
- 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
Do not discuss
- Cesium chloride -- getting very clean DNA, ethidium used
- Banding DNA
Demonstration
- Centrifugation demonstration -- either the arabidopsis sample or the DNA sample
Intruigue
- Sodium Acetate vs NaCl in precipitation, Kinases dislike Na+ -- why or why not?