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biochem > lab techniques > Flashcards

lab techniques Flashcards

1
Q

gel electrophoresis separates what

A

macromolecules (proteins, DNA, RNA)

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2
Q

what is used for proteins/small molecules in gel electrophoresis

A

polyacrylamide

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3
Q

what is used for larger molecules in gel electrophoresis

A

agarose

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4
Q

negatively charged molecules travel toward

A

anode at bottom

in gel electrophoresis

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5
Q

larger molecules move…

A

SLOWER in gel electrophoresis

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6
Q

what dye is used in gel electrophoresis

A

coomassie blue stain for visualization

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7
Q

native-PAGE is a method where

A

polyacrylamide gel electrophoresis method for proteins is utilized with NON-DENATURING conditions

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8
Q

proteins keep their native charge and structure so that…

A

they are separated based on charge and size

in native-PAGE

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9
Q

in SDS-page, a polyacrylamide gel electrophoresis method is used for

A

proteins using DENATURING conditions

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10
Q

sodium dodecyl sulfate denature the…

A

proteins and give them a uniform charge

part of SDS-PAGE

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11
Q

proteins separate solely on mass and then you can…

A

estimate the protein’s molecular mass

SDS-PAGE

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12
Q

reducing SDS-PAGE is the same as

A

SDS-PAGE but adds a reducing agent

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13
Q

reducing agent in reducing SDS-PAGE will reduce

A

disulfide bridges and result in completely denatured protein

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14
Q

isoelectric focusing is where a gel electrophoresis method is used to

A

separate proteins on the basis of relative contents of acidic/basic residues

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15
Q

gel has pH gradient and proteins will migrate through gel until….

A

they reach pH that matches isoelectric point

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16
Q

at the pI, the protein has a neutral charge so

A

it will no longer be attracted to the anode and it will stop migrating

part of isoelectric focusing

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17
Q

southern blotting

A

detection of a specific DNA sequence in a sample

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18
Q

northern blotting is the detection of

A

specific RNA sequences in a sample

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19
Q

western blotting is the detection of a

A

specific protein in a sample

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20
Q

sanger DNA sequencing uses what method

A

chain termination method

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21
Q

chain termination method uses

A

dideoxy nucleotides

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22
Q

ddNTP lacks a

A

hydroxyl group on 3’ carbon on sugar ring

no more nucleotides can be added to the chain w/ the 3’ hydroxyl group missing

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23
Q

chain ends with the

A

ddNTP that is marked with a particular color of dye depending on base carried

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24
Q

polymerase chain reaction (PCR) is used to make

A

many copies of a specific DNA region in vitro

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25
Q

key ingredients of PCR =

A

Taq polymerase

primers

template DNA

nucleotides (DNA building blocks)

26
Q

ingredients are assembled in a tube along with cofactors needed by the enzyme and put through…..

A

repeated cycles of heating/cooling that allow DNA to be synthesized

27
Q

primer must have high…

A

GC content and either a G or C at each end

28
Q

enzyme used in PCR

A

taq polymerase

29
Q

taq polymerase is named after the

A

heat-tolerant bacterium from which it is isolated

very heat-stable and most active around 70 C

30
Q

steps of PCR

A

1) denaturation (96 C)

2) annealing (55 - 65 C)

3) extension (72 C)

31
Q

PCR’s cycle is repeated until

A

you have enough DNA

32
Q

chromatography separates

A

two or more molecules from a mixture

33
Q

stationary phase is

A

typically polar

34
Q

polar molecules elute

A

SLOWER

35
Q

mobile phase is

A

typically nonpolar

36
Q

nonpolar molecules elute

A

faster

37
Q

liquid chromatography utilizes

A

silica in stationary phase

toluene/nonpolar liquid used as mobile phase

38
Q

HPLC is a type of liquid chromatography that uses high pressure to pass…

A

the solvent phase through a more finely-group stationary phase

increases interactions b/w molecules and stationary phase

39
Q

high-performance liquid chromatography (HPLC) has

A

higher resolving power

40
Q

gas chromatography vaporizes liquid before

A

separation

41
Q

molecules are separated based on

A

polarity and boiling point

42
Q

stationary phase in gas chromatography is a

A

thin layer of material applied in column’s inside

43
Q

the polarity of the stationary phase matches that…

A

of the solute in gas chromatography

44
Q

mobile phase of gas chromatography is

A

an inert gas

45
Q

size-exclusion chromatography separates molecules by

A

size rather than polarity

46
Q

smaller molecules enter the porous gel beads, allowing them to

A

elute later

47
Q

larger molecules elute faster because

A

they do not fit in the pores and will not be slowed down

48
Q

ion-exchange chromatography separates

A

proteins by net charge

49
Q

column is filled with

A

charged beads either positive or negative

part of ion exchange chromatography

50
Q

cation exchange uses

A

negative beads

negative proteins elute first

51
Q

anion exchange uses

A

positive beads

positive proteins elute first

52
Q

affinity chromatography separates proteins based on

A

their affinity for a specific ligand

53
Q

beads are bound to a specific ligand and proteins with a…

A

high affinity for that ligand will bind to the beads

affinity chromatography

54
Q

proteins with a low affinity for the

A

ligand with elute first in affinity chromatography

55
Q

thin-layer chromatography is a

A

sheet coated in polar silica gel

56
Q

molecules are spotted at the

A

bottom of the sheet

thin-layer chromatography

57
Q

sheet is placed in a nonpolar liquid and the mobile phase…

A

travels up the plate using capillary action

58
Q

nonpolar molecules have the highest

A

Rx value

59
Q

retention factor (Rf) =

A

distance spot moved / distance solvent front moved

60
Q

central dogma

A

DNA –> transcription –> RNA –> translation –> PROTEIN

61
Q
A

biochem (11 decks)

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