help molecular bio question (1 Viewer)

[dronee]

You have a dye, Sybr GreenI which binds only to double stranded DNA (not single stranded). Once bound, it fluoresces strongly and can be used to monitor DNA melting (transition from double stranded to single stranded).

(i) on the axes provided draw the DNA melt curve for a sample of E. coli DNA which has been mixed with Sybr GreenI.
--> the axes shown has Temperature for x-axis and Fluorescence for y-axis.
on the same graph show the melt curve for a sample of DNA
(ii) with a lower %(G+C) content
(iii) from the same source dissolved in a buffer at pH>9 prior to the measurements.


my attempt at answering:
(i) okay so im thinking the shape of the curve is like an S
kind of like this: http://www.entelechon.com/wp-content/uploads/2008/10/dna_melting-300x178.gif
but is it exactly like that? or is the other way?

(ii) for this one i know that i just have to draw the exact same shape except more towards 0 since more GC content means higher melting point.

(iii) im not sure about this one.
help please?

 

[jet]

You have to think about what is actually happening. The Sybr GreenI is binding to double-stranded DNA and fluorescing. You're measuring the fluorescence.

Then, you slowly heat up the mixture and as you heat it up, the H-bonds in the DNA will begin to break and slowly the strands will come apart. When they come apart, the Sybr GreenI has to drop off and it will stop fluorescing.

So, how much fluorescence would you see at a low temperature and how much would you see for a high temperature?

 

[dronee]

You have to think about what is actually happening. The Sybr GreenI is binding to double-stranded DNA and fluorescing. You're measuring the fluorescence.

Then, you slowly heat up the mixture and as you heat it up, the H-bonds in the DNA will begin to break and slowly the strands will come apart. When they come apart, the Sybr GreenI has to drop off and it will stop fluorescing.

So, how much fluorescence would you see at a low temperature and how much would you see for a high temperature?

ohh, that makes sense!