Test 1: Nucleic acid detection

Question 1

Why do nucleotides absorb UV radiation?

Answer 1

Aromatic purines and pyrimidines

Question 2

At what wavelength in UV is the absorption maximum for DNA and RNA?

Answer 2

260

Question 3

1 optical density unit =
__ ug/ml for dsDNA
__ ug/ml for RNA
Why?

Answer 3

50
40
Base pair stacking results in reduced absorption intensity, so more are needed to absorb 1 optical density unit.

Question 4

EtBR stands for what?

Answer 4

Ethidium bromide

Question 5

Ethidium bromide:

Answer 5

Intercalates between bases, usually in DNA but sometimes RNA, unwinding them and increasing the pitch.
It absorbs UV and fluoresces bright red.
Its florescence is 20 - 30x stronger when when binding bases

Question 6

What is an intercalating fluorophore?

Answer 6

Substance which slips between bases, and in doing so, increases its florescence intensity.

Question 7

Cesium Chloride (CsCl) centrifugation:

Answer 7

CsCl when centrifuged sets up a density gradient. RNA can be seperated from dsDNA and ssDNA, because RNA will normally pellet.
This is equilibrium centrifugation.

Question 8

What is the typical density for:
dsDNA
ssDNA
RNA

Answer 8

1. 71
2. 72
3. 79

Question 9

This is the formula for dsDNA density
density = 1.66 + .001(%G+C) (unit is g/ml)
What does it imply?

Answer 9

G:C pairings either compact the helix, or they weigh more with the same type of compaction.

Question 10

Rate-Zonal Centrifugation

Answer 10

Sample centrifuged through gel or other thick substance. Separated by density due to movement rate through gel.

Question 11

Equilibrium Centrifugation:

Answer 11

A density gradient exists in the fluid (like CeCl), the sample is centrifuged until equilibrium, with denser samples settling closer to the bottom.