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RDT_CDW > Characterization of DNA molecules > Flashcards

Characterization of DNA molecules Flashcards

1
Q

How DNA is isolated?

A

disrupt membrane - add lysozyme or detergent

add alcohol

dna precipitates at the interface

pick up using a glass rod and transfer to aqua tube

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2
Q

Methods to characterize dna

A

by size - gel

by order of N bases - sequencing

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3
Q

Seperation by gel depends on?

A

charge

size

shape

ionic nature

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4
Q

Give the equation for migrating velocity? mobility?

A

v = zeE/f

mobility = Ze/f

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5
Q

What is mobility?

A

distance of each band from origin with respect to migration front distance

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6
Q

What are factors affecting the movement of molecules?

A

Charge - direction

voltage applied - relative velocity

shape & size

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7
Q

What are the steps in electrophorosis? and show how each is achieved?

A

induce a charge to mol

prepare a inert matrix

allow the molecule to run

locating

determining size

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8
Q

What are the matrix substances used for DNA & protein?

A

agarose

polyacrylamide

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9
Q

Short note for agarose? agarose 3d structure?

A

heat - cross-links are broken

cool - cross-links reform

galactan

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10
Q

Acrylamide gel characteristics? speciality? Drawback?

A

small cavities - small can be identified (one bp diff.)

hard - O2 inhibit the polymerization

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11
Q

What are the challenges in electrophoresis?

A

dna should run thru the gel

deposition of analyte in the well

finding the gel front

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12
Q

What are used as the gel running buffer? components and their function?

A

TAE or TBE

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13
Q

Loading dye components and their functions? functions of a loading dye?

A

sucrose/glycerol - dna sink in to the well

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14
Q

Parts of electrophoresis equipment?

A

ss

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15
Q

How conformation of DNA affects gel mobility?

A

supercoiled - fastest

supercoiled denatured

relaxed circular

linear

nicked open circular(slowent)

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16
Q

How to detect DNA?

A

eth br intercalates with the bases of DNA – UV – fluorescent glow

17
Q

What is the relationship bw DNA MW and distance travelled?

A

inverse for log of MW

18
Q

Draw fingerprinting standard curve. mark the deviation at large sizesp

A

ss

19
Q

For what SDS PAGE is used? properties? components? common stains?

A

vertical gel

to mw – proteins

acrylamide + SDS

  • coomassie blue
  • copper stain
  • silver stain
20
Q

List the main DNA sequencing methods

A

maxam gilbert

sanger

next gen

21
Q

Explain the dideoxy method. depends on?

A

ss

22
Q

Sanger method: principles?

requirements?

A 
template dna 
primer
thermostable pol 1
dNTPs & ddNTPs* tagged
terminator
23
Q

Sanger method: what are the 4 rxns to run?

A

ddATP, ddCTP, ddGTP, ddTTP

24
Q

How to detect the oligonucleotide & build sequence?

A

ss

RDT_CDW (10 decks)

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