Week 12: Western Blots and PCR Flashcards
Week 12
In a western blot, what are the three main things that are required?
Antibody
Transfer chamber
Detection method
Week 12
In a western blot, what does an antibody need?
A protein or epitope tag
Week 12
What are the main purposes of a western blot? (2)
Protein tracking during purification
Determining regulation
during cell cycle/growth.
Week 12
What question does quantitative western blot analysis answer?
How much is the protein being expressed at a given point in the cell cycle?
- Westerns are useful in confirming the expression of proteins *
Week 12
Endpoint PCR (aka General PCR) is generally limited to detection of what?
genetic potential
Week 12
When designing primers, it is important to reminder the wobble code. What is this?
More than one nucleotide can code for a protein
Week 12
What is reverse transcriptase PCR used for?
Used to detect when during cell growth or the cell cycle genes are expressed ( timing )
Used to detect transcription when environmental conditions change.
Week 12
Multi-plex PCR is used as a rapid identification method. Why is this?
Requires MULTIPLE primer sets ( ran at the same time ), specific to the target organism
Week 12
What are the steps for designing a multi-plex system? (3)
1) Select target genes and design primers
Primers must be temperature matched and each set should yield a different sized fragment ( base count )
2) Test primers for selectivity — Amplify individual amplicons and check via sequence analysis that you have the correct target
3) Test primers in all-in-one situation. If this works, test under conditions envisioned for the organism.
Week 12
What is the housekeeping gene for Vibrio sp.?
recA
sometimes omp
Week 12
How are Vibrio sp. and PCR connected?
Toxigenic Vibrio sp. can carry multiple toxin genes.
Toxins can also vary between species, providing the opportunity to develop PCR systems that can detect multiple potential threats from a single sample
