Assignment 2 questions. Flashcards
DNA quality and quantity assessments are critical prior to determining what types of genetic tests would be most appropriate for the sample. A260/280 can be performed on a Nanodrop machine. From a fresh muscle tissue sample of about 10mg, your extraction yields an A260nm absorption reading that corresponds to about 5ng/ul yield in a 200ul elution and an A260/280 ratio of 1.95. What can you say about the success of your extraction?
yield of 5ng/ul in 200ul is in the approximate range expected of a tissue sample of 10mg
What is the purpose of the final extension step at 60°C for 45 min during PCR?
To ensure all sequences have a non-template addition of an A at the end of the product
describe FIVE controls you would employ for the extraction and amplification steps of this work and what each control might consist of/contain.
Extraction neg, extract pos, amp neg, amp pos, amp primer control
T/F: During qPCR, a relative increase in fluorescence is only observed for a sample when the quencher is separated from the MGB.
false, the quencher does not get separated it is the reporter
You are tasked with designing new primers for the species-specific DNA amplification of the invasive aquatic plant, water hyacinth, from water samples looking to assess for early invasions of this plant. You decide that cpDNA would be the best target given the higher copy numbers of this genome in plants. As you look through online genetic databases for regions of the chloroplast genome in this species appropriate for primer design, what is required in ANY primer pair design needed to produce a species-specific and locus-specific DNA amplicon?
the primer is long enough, based on the size of the genome, to most likely only anneal to one region of the genome
the primer is short enough to avoid a melting temperature too close to the extension temperature
the primer is assessed for its likelihood to anneal to other species based on a BLAST search of the primer sequence
What is the difference between Sybr Green and TaqMan when running quantitative PCR (qPCR)?
sybr green is less specific
Provide two reasons why want Mg2+ in a PCR after removing it from DNA extracts using chelating agents.
Taq cofactor
primer specificity
You are screening for black bear DNA in a facial cream being marketed as containing bear grease and bear gall given their putative regenerative properties. Selling such a product, if there is bear DNA in it, would be illegal. You use qPCR, but you only get very low levels quantified, basically at the threshold of detection for the assay relative to your standard curve of known amounts of black bear DNA. what might be concerns to consider when reporting on your findings?
The samples being tested could contain other closely related species that might cross-amplify.
The R squared value is < 0.95 suggesting your standard curve might not allow you to clearly assess the amount of DNA in the sample.
The PCR reaction was running at 80% efficiency vs the ideal doubling of product every cycle, and thus might not allow you to clearly asses the amount of DNA in the sample.
