Minutes 20101101 TA BIOL 208 - PCR etc.
From SnOwy - Ed's Wiki Notebook
Contents |
Topics this week
- Southern Blotting
- PCR
- DNA Sequencing
- all of these topics appear on the quizzes
Southern Blotting
- stringency -- specificity of the method
- why before genomic sequencing?
- allows identification of correct probe
PCR
- stringency -- specificity of the method (primers / annealing temperature)
- which sequence do we order when we want a primer (Fwd, Rev)
- adding restriction sites -- modified primers
- sequencing a PCR product
- allows us to ensure that we have the in-frame
Sequencing
- ladder
- tie it back to a gel
- 5' to 3' -- sending a strand off to sequencing -- where is the sequence with respect to the gel
- don't go into detail for pyrosequencing -- it exists
- light on Maxam-Gilbert "used for DNA footprinting" -- almost end of story
- should understand how to read a Maxam-Gilbert gel
- explain dideoxy-sequencing
Rough Parts
- sequencing genomes -- don't discuss
- primer walking -- discuss
Positive and Negative Controls
Southern Blotting
- southern blotting -- rarely do a negative control -- only if we're testing if the organism has a transgene
- in general, just looking at a genome and asking if something is there
- give a scenario -- positive controls -- see UWACE
- questions for controls -- what positive control is used for a specific oligo? Complementary sequence
- probing a southern with a riboprobe, what positive control? The template used to make it
PCR
- screening -- positive -- use something we know always works
- positive control not possible in colony PCR -- primers
- things don't always work
Cover this week
- Why controls are important
- Southern Blotting -- Focus on Specificity
- PCR -- Focus on Specificity
- Primer Design emphasis
- Why shorter, why longer
- DNA Sequencing -- Dideoxy Only
