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MOL. BIO LAB > MIDTERM LAB: ELECTROPHORESIS > Flashcards

MIDTERM LAB: ELECTROPHORESIS Flashcards

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Biology 101 flashcards
1
Q

Most practical and recently use of resolution & detection of nucleic acids
- Separation of molecules in electric current.

A

ELECTROPHORESIS

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2
Q

ability to distinguish separate individuals components of nucleic acids.

A

RESOLUTION

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3
Q

ELECTROPHORESIS Purpose: Separate molecules by ?

A

SIZE AND CHARGE

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4
Q

The negative pole.

A

CATHODE

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5
Q

POSITIVE POLE

A

ANODE

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6
Q

NEGATIVELY CHARGED MOLECULE

A

ANION

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7
Q

POSITIVELY CHARGED MOLECULE

A

CATION

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8
Q

anions travel to the ?

A

anode

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9
Q

cations travel to the ?

A

cathode

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10
Q

Nucleic Acids (NA) moves from

A

NEGATIVE TO POSITIVE POLE

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11
Q

When DNA is applied to a macromolecular case or gel, its
migration under the pull of current is

A

IMPEDED

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12
Q
  • May move rapidly in large pore size
    (Low gel concentration)
  • May move slowly in small pore size
    (High gel concentration)
A

LARGE NUCLEIC ACID FRAGMENTS

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13
Q
  • May move rapidly in large pore size
    and small pore size.
A

SMALL NUCLEIC ACID FRAGMENTS

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14
Q
  • to separate SMALL NUCLEIC ACID FRAGMENTS, it should be in______________(pores become small)
A

HIGH GEL CONCETRATIONS

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15
Q

The CONCENTRATION OF GEL/BUFFERS will affect the _____ of fragments of different size ranges.

A

RESOLUTION

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16
Q

sample are spotted in the center of the paper, high voltage is applied and the spots migraine according to their
charges

A

PAPER

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17
Q

separates AMPHOTERIC molecules (have both acidic & basic properties/alkaline properties (protein)) according to their charged as defined by the pKa values of proton-accepting sites within a molecule

A

TUBE (ISOELECTRIC FOCUSING)

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18
Q
  • most frequently used
  • horizontal/vertical
  • sample is introduced into the gel at
    the cathode end and migrates with the current toward the anode
A

SLAB GELS

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19
Q

separation technique of molecules and ions performed in a narrow capillary tube structure using electric current

A

CAPILLARIES

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20
Q

Provide resistance to the movement of molecules under the force of electric current forming bands.(DNA fragments)

A

GEL SYSTEMS

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21
Q
  • The movement is impeded in the gel and will form it.
    o Dependent to the speed of migration.
A

BANDS

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22
Q

Serve as support medium for analysis of the separated components

A

GEL

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23
Q

As the gel concentration increases, the movement of particles (DNA fragments) ______

A

SLOWS

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24
Q

CRITERIA FOR A GOOD GEL:

A
  1. Unaffected by electrophoresis
  2. Simple to prepare
  3. Amenable to modification
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25
Q

TYPES OF GEL:

A
  • AGAROSE
  • POLYACRYLAMIDE
  • COMPOSITE AGAROSE-POLYACRYLAMIDE
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26
Q
  • Polysaccharide polymer of AGAROBIOSE
  • Is composed of D - galactose
  • Extracted from SEAWEEDS
  • Component in agar
  • Usage: Powder is suspended in buffer, heated and poured into mold.
  • Cooled 55 – 56 °C
  • Formulations:
    o Small DNA (50-500 BP) – 2-3%
    o Large DNA(2000 – 5000 BP)
  • Easier casting
  • Gel is NEGATIVELY CHARGED – thus surrounded by buffers
  • Usually on HORIZONTAL SETUPS
A

AGAROSE GEL

27
Q
  • SYNTHETIC
  • Repeating units: Acrylamide + methylene bisacrylamide
  • Higher resolution than agarose
  • Mobility can depend on DNA sequence (agarose do not, solely on size only.
  • For very small DNA,SSDNA, RNA, proteins
  • Forms: Powdered form (neurotoxic), commercial solutions, and performed gels.
  • Usually in VERTICAL setups
  • Requires at polymerization catalysts (for gel to solidify)
A

POLYACRYLAMIDE GEL

28
Q

POLYACRYLAMIDE GEL Requires at polymerization catalysts (for gel to solidify) such as?

A
  • AMMONIUM PERSULFATE
  • TETRAMETHYLENEDIAMINE
  • LIGHT ACTIVATION
29
Q
  • Carry the current and protect samples during electrophoresis.
  • Composed of weak acid+ its conjugate base
  • Uncontrolled ph may damage the sample
  • High concentration means higher conductivity, thus we need to lower the voltage.
A

BUFFERS

30
Q

BUFFERS FOR DNA?

A
  1. TRIS BORATED EDTA (TBE)
  2. TRIS ACTETATE EDTA (TAE)
  3. TRIS PHOSPHATE EDTA (TPE)
31
Q

BUFFERS FOR RNA

A
  1. 10nM SODIUM PHOSPHATE
  2. MOPS BUFFER
32
Q

BUFFER ADDITIVES THAT BLOCK HYDROGEN BONDING SITES

A

FORMAMIDE + HEAT

33
Q

BUFFER ADDITIVES THAT PREVENT FOLDING

A

UREA + HEAT

34
Q

__________prevents hydrogen bonds and formation of HETERODUPLEXES – FOLDED SSDNA/RNA forming HAIRPIN-LIKE structures that will impair MIGRATION.

A

DENATURING AGENTS

35
Q

 Gel is positioned horizontally
 Gel is submerged throughout the loading and electrophoresis process
 Samples are located into wells at one end of the gel

A

HORIZONTAL/SUBMARINE GEL (AGAROSE GEL are commonly used)

36
Q

 Gel is positioned vertically
 Samples are loaded into wells at the TOP OF THE GEL
 Spacers: determine the thickness of the gel

A

VERTICAL GEL (POLYACRYLAMIDE GEL
are often used)

37
Q

 used to create wells in the gel where sample can be loaded

A

ELECTROPHORESIS COMBS

38
Q

determine the CAPACITY of the well for the sample

A

SIZE OF TEETH

39
Q

determine the number of wells

A

NUMBER OF TEETH

40
Q

TYPE OF ELECTROPHORESIS COMB:
-WELLS SEPARATED BY AN “EAR” OF THE GEL.
- placed on TOP of the gel.
- evenly spaced teeth = creates well of uniform size & is suitable for general purposes; DNA/protein electrophoresis

A

REGULAR COMBS

41
Q

TYPE OF ELECTROPHORESIS COMB:
-WELLS IMMEDIATELY ADJACENT
-Placed upside-down, after polymerization Is placed side-down at top.
- teeth resembling a pointed/shark’s tooth
- for preventing sample leakage/ improving loading accuracy

A

HOUNDSTOOTH/SHARKSTOOTH

42
Q

RUNNING A GEL

A

➢ Use the proper gel concentration
for sample size range
➢ Use the proper comb (well) and
size
➢ Load sample mixed with: TRACKING DYE & DENSITY AGENT

43
Q

to monitor the progress of the electrophoresis run.(Migrate at specific speeds in a given gel concentration.)

EX: bromophenol blue; xylene cyanol green

A

TRACKING DYE (dye + density agent)

44
Q

allows nucleic acids to fall in the sample wall. (Allow sinking of the nucleic acid to the wall instead of diffusing to the buffer.)

EX: sucrose, ficoll, glycerol

A

DENSITY AGENT

45
Q

Action of putting the isolated nucleic acid in wells.

A

GEL LOADING

46
Q

Prior to loading, THINGS SHOULD
BE ADDED:

A
  • Tracking dyes
  • Density agent
47
Q

ELECTROPHORESIS WILL TERMINATE IF:

A

 DYE APPROACHES TO THE END OF
THE GEL, OR
 OBTAINED THE DESIRED DISTANCE

48
Q

 Visualization of bands after electrophoresis

A

DETECTION SYSTEMS

49
Q

to make the separated molecules
visible

A

STAIN

50
Q

For visualization of bands:

A
  • UV TRANSILLUMINATOR
  • FLUORESCENCE IMAGING SYSTEM
  • GEL DOCUMENTATION SYSTEM
51
Q

COMMONLY USED STAINS

A
  • FLUORESCENT DYES
  • SILVER STAINS
52
Q

FLUORESCENT DYES:
stack (intercalate) between adjacent nitrogen based in double stranded nucleic acids.

A

INTERCALATING AGENTS

53
Q

FLUORESCENT DYES:
sits in the minor groove of the double helix

A

Minor groove – binding dyes

54
Q

Preferred for real time PCR

A

SYBR GREEN

55
Q
  • originally developed for protein
    visualization
     Fixed with methanol + acetic acid
     insoluble in black silver after formaldehyde
A

SILVER STAINS

56
Q

SILVER STAINS 2 METHODS

A
  • SILVER DIAMINE/AMMONIACAL SILVER
    -SILVER NITRATE
57
Q
  • For very large DNA fragments (50k
    to 250 + kBP)
  • Pulses of current applied to the gel
    in alternating dimensions enhance
    migration.
A

PULSE-FIELD GEL ELECTROPHORESIS

58
Q

PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Alternating positive and negative poles, provides good resolution (>800kb)

A

FIELD INVERSION GEL ELECTROPHORESIS (FIGE)

59
Q

PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Transverse angle reorientation of
poles in a vertical gel.

A

TRANSVERSE ALTERNATIVE GEL
ELECTROPHORESIS (TAFE)

60
Q

PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Alternating polarity in an electrode
array; DNA molecules as large as 2Mb can be well separated.

A

CONTOUR- CLAMPLED HOMOGENOUS
ELECTRICAL FIELD (CHEF)

61
Q

PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Rotating gel with fixed poles; 50kb to
6000 KB

A

ROTATING GEL ELECTROPHORESIS
(RGE)

62
Q

PULSE FIELD GEL ELECTROPHORESIS APPLICATIONS

A

 Molecular typing and identification of pathogens
 Gold standard method of some bacterial ids
 Genotyping for antibiotics resistance of:
o Staphylococcus
aureus
o Pseudomonas
aeruginosa
o Acinetobacter
baumannii
o Mycobacterium
tuberculosis
 Epidemiological studies
 Relationship of strains of same
 DNA fingerprinting of viruses

63
Q

 Separates solutes by charge/mass ratio
 Separated nucleic acids,
metals, anions, carbohydrates, pharmaceuticals
 Thin glass (fused silica)
capillary 30-100cm X 25-100um
internal diameter
 Linear or cross-linked polyacrylamide or other linear polymers used for sieving
 Separation based on size

A

CAPILLARY ELECTROPHORESIS

MOL. BIO LAB (9 decks)

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