Detecting, Quantifying & Resolving Nucleic Acids Flashcards Preview

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Flashcards in Detecting, Quantifying & Resolving Nucleic Acids Deck (32)
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1

methods for DNA quantification

UV spectroscopy
Fluorometry
gel electrophoresis

2

Absorbance spectroscopy

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

3

Fluorescence spectroscopy

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

4

UV wavelengths

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

5

DNA maximal absorption

at 260 nm

6

dsDNA 1/extinction coefficient

50

7

RNA 1/extinction coefficient

40

8

ssDNA 1/extinction coefficient

33

9

DNA concentration calculation

absorbance @ 260 x 1/coefficient x dilution

10

most common DNA contaminant

proteins

11

maximal absorption of proteins

280 nm

12

260/280 ratio

>1.6 to be able to use in downstream reactions
measure of protein contamination

13

Nanodrop

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

14

Hoechst 333342/33258

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

15

fluorescent RNA binding dye

PicoGreen or
RiboGreen

16

fluorometry pros & cons

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

17

Gel electrophoresis principle

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

18

gel electrophoresis can determine

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

19

DNA charge

negatively charged due to the phosphodiester backbone

20

agarose gel

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

21

0.8% agar

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

22

2.0% agar

75-500 bp resolution
small PCR products

23

TAE

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

24

TBE

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

25

TAE & TBE functionality

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

26

Ethidium Bromide

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

27

SYBR green or SYBR gold

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

28

PAGE

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

29

Capillary electrophoresis

separates solutes by charge/mass ratio
more rapid, automated than slab gels

30

RNA electrophoresis problem

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

31

RNA electrophoresis

PAGE for sequencing/STR only (high resolution)

32

Automation

principles of electrophoresis & fluorescence