DNA Quantitation Part 2 (Exam 2 & 3) Flashcards
For agarose gel quantitation, what does the endpoint measurement check for?
Band intensity
For rtPCR quantitation, the end product is a…
Standard (amplification) curve
In rtPCR, what is the relationship between the quantity of DNA and number of cycles observed?
Increase DNA quantity (ng) = Decrease in # of cycles
In rtPCR, what is the relationship between copy number, threshold, and cycle number?
More copies = amplification curve reaching threshold in fewer cycles
The level of detection or point at which a reaction reaches a fluorescent intensity above the base line.
What is the threshold?
The cycle at which a sample’s amplification curve crosses the threshold.
What is the threshold cycle?
What are the 3 types of electrophoresis?
1) Agarose
2) Acrylamide
3) Capillary
How is information collected by rtPCR?
Automatically on a cycle-by-cycle basis
Given that the copy number in PCR doubles on each cycle, what graphical function to be used to describe it (HINT: f(x) = …)?
f(x) = 2^x
What are 2 advantages of PCR?
1) Small, degraded amount of DNA can be used
2) Non-human DNA does not interfere
What are 2 disadvantages of PCR?
1) PCR inhibition
2) External DNA can easily contaminate
In 310 capillary electrophoresis, the amplified fragments are separated by { } and { }.
Color & Size
What is the purpose of laminar hood flow in PCR?
To prevent contamination
What are 2 advantages of RFLP?
1) No contamination issues
2) No expensive equipment
What are 2 disadvantages of RFLP?
1) Lot of DNA needed (~100 ng)
2) Good quality DNA needed
