Detecting, Quantifying & Resolving Nucleic Acids

Question 1

methods for DNA quantification

Answer 1

UV spectroscopy
Fluorometry
gel electrophoresis

Question 2

Absorbance spectroscopy

Answer 2

goal is to measure the light of a specific wavelength absorbed by an analyte (chromophore)

Question 3

Fluorescence spectroscopy

Answer 3

light emitted from an analyte (fluorophore) following the excitation by a light of a specific wavelength is measured

Question 4

UV wavelengths

Answer 4

180-350 nm in length
used for nucleic acids
cannot use plastic cuvettes bc they will absorb this wavelength range

Question 5

DNA maximal absorption

Answer 5

at 260 nm

Question 6

dsDNA 1/extinction coefficient

Answer 6

50

Question 7

RNA 1/extinction coefficient

Answer 7

40

Question 8

ssDNA 1/extinction coefficient

Answer 8

33

Question 9

DNA concentration calculation

Answer 9

absorbance @ 260 x 1/coefficient x dilution

Question 10

most common DNA contaminant

Answer 10

proteins

Question 11

maximal absorption of proteins

Answer 11

280 nm

Question 12

260/280 ratio

Answer 12

> 1.6 to be able to use in downstream reactions

measure of protein contamination

Question 13

Nanodrop

Answer 13

small UV spectrophotometer
uses only 1 uL of DNA
determines concentration & purity
no dilutions
does not tell about the quality of the DNA

Question 14

Hoechst 333342/33258

Answer 14

fluorescent DNA binding dye
absorbance maximum 350 nm
emission maximum 461 nm (blue)

Question 15

fluorescent RNA binding dye

Answer 15

PicoGreen or

RiboGreen

Question 16

fluorometry pros & cons

Answer 16

pros: very quantitative, not influenced by contamination
cons: dyes are instable in light, says nothing about integrity of sample

Question 17

Gel electrophoresis principle

Answer 17

negatively charged molecule will migrate towards the anode (+)
migration depends on size, charge & shape

Question 18

gel electrophoresis can determine

Answer 18

presence of nucleic acid
purity
INTEGRITY
quantify
measure size/ length!

Question 19

DNA charge

Answer 19

negatively charged due to the phosphodiester backbone

Question 20

agarose gel

Answer 20

polysaccharide purified from seaweed
used as a molecular sieve
varying concentrations determine the resolution of the fragments of DNA

Question 21

0.8% agar

Answer 21

500-3000 bp resolution
assessing genomic DNA integrity
large DNA fragments

Question 22

2.0% agar

Answer 22

75-500 bp resolution

small PCR products

Question 23

TAE

Answer 23

tris-acetate EDTA
cheap, easy to use
effective for running larger sized DNA fragments

Question 24

TBE

Answer 24

tris-borate EDTA
2x buffering capacity of TAE
resolves small fragments better (

Question 25

TAE & TBE functionality

Answer 25

both buffers bind divalent cations such that DNA has a uniform negative charge & runs strictly according to size

Question 26

Ethidium Bromide

Answer 26

most commonly used agarose gel electrophoresis stain
added directly to the gel
sensitivity ~10ng of DNA
possible carcinogen

Question 27

SYBR green or SYBR gold

Answer 27

gel electrophoresis stain
cannot add directly to gel
takes 30 min to stain
sensitivity ~1ng DNA
not toxic
not used

Question 28

PAGE

Answer 28

polyacrylamide gel electrophoresis
synthetic, consistent polymer
resolves 1 bp difference in a 1kb molecule

Question 29

Capillary electrophoresis

Answer 29

separates solutes by charge/mass ratio

more rapid, automated than slab gels

Question 30

RNA electrophoresis problem

Answer 30

RNA has a high degree of secondary structure & will not run in an electric field according to its length unless it is denatured

Question 31

RNA electrophoresis

Answer 31

PAGE for sequencing/STR only (high resolution)

Question 32

Automation

Answer 32

principles of electrophoresis & fluorescence