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[Molecular Biology] Lec > LESSON 9 GEL ELECTROPHORESIS > Flashcards

LESSON 9 GEL ELECTROPHORESIS Flashcards

1
Q

Technique used for the separation, detection, visualization and sometimes purification of biomolecules DNA, RNA and protein using an electric field applied to a gel matrix

A

gel electrophoresis?

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2
Q

A technique used to separate DNA fragments according to their size.

A

gel electrophoresis?

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3
Q

Purpose gel electrophoresis It is used to visualize, measure the size of the DNA fragments and even identify contaminants from the
[?]: millions of DNA produced
[?]: to check if you will be “pasting” the correct gene

A
  1. PCR test
  2. DNA cloning/ Recombinant technology
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4
Q

Sample has already run thru the electric current and have migrated from the

A

negatively charged(cathode) to the positively charged (anode) area

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5
Q

: diabetic patients (9 patients)

A

TGF-B gene

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6
Q

o standard/reagent/DNA ladder

A

M (molecular weight markers)

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7
Q

o serves as a basis for the length of DNA with a unit of “bp” (base pair)

A

M (molecular weight markers)

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8
Q

= 1 kbp

A

1,000 bp

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9
Q

: DNA ladder

A

Lane 1

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10
Q

: has a DNA fragment of 1,500 bp

A

Lane 2

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11
Q

: has a DNA fragment of 600 bp (with TGF-B gene)

A

Lane 3-9

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12
Q

: fluorescent dye stain

A

Ethidium bromide

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13
Q

Used for visualization under UV light/transilluminator

A

Ethidium bromide

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14
Q

To get the fragment (?), elute or cut the agarose gel (DNA portion) → wash

A

600 bp

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15
Q

acts as a strainer for the separation of particles

A

gel in electrophoresis

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16
Q

: fastest to migrate

A

Small DNA/RNA fragemnts/shorter base pairs

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17
Q

voltages involved

A

(positive and negative charges)

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18
Q

separation according to

A

size of bp

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19
Q

2 kbp from 10 bp =

A

2 kbp gene

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20
Q

DNA fragments are [?] (loaded at the cathode), so they move towards the positive electrode (anode).

A

negatively charged

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21
Q

presence of phosphate group (phosphoric acid)

A

negatively charged

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22
Q

Separation takes

A

30-45 mins

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23
Q

: DNA fragments will overpass

A

Long electrical runs

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24
Q

Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel [?] than large ones.

A

faster

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25
Q

Voltage:
Depends on the protocol

A

60-120 V

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26
Q

Separated in [?]; test is optimized; proper migration to properly identify their sizes

A

bands

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27
Q

REAGENTS EQUIPMENTS AND REAGENTS/ELECTROPHORESIS SYSTEM

A

Power supply and chamber
Gel casting tray/gel box/ gel chamber:
Gel comb:
Buffer dam or tapes:

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28
Q

a source of power supply

A

Power supply and chamber

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29
Q

: hold/mold the agarose

A

Gel casting tray/gel box/ gel chamber

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30
Q

: create holes or sample wells in the gel where the samples are loaded

A

Gel comb

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31
Q

: limits the movement of the agarose gel

A

Buffer dam or tapes

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32
Q

Matrix where samples are loaded and DNA migrates

A

Agarose

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33
Q

Commonly used in gel electrophoresis

A

Agarose

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34
Q

For big DNA fragments from 50-20,000bp

A

Agarose

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35
Q

High resolving power for short 5-500bp

A

Polyacrylamide

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36
Q

Horizontal

A

Agarose

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37
Q

Vertical

A

Polyacrylamide

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38
Q

High agarose conc: smaller pore size

A

Agarose

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39
Q

Routinely 6-15%

A

Polyacrylamide

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40
Q

Set as it cools

A

Agarose

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41
Q

Sets by chemical reaction once crosslinking occurs

A

Polyacrylamide

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42
Q

Polysaccharide extracted from sea weed

A

Agarose

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43
Q

Crosslinked polymer of acrylamide
-Neurotoxin
-P.A.: nontoxic

A

Polyacrylamide

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44
Q

Easy to prepare

A

Agarose

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45
Q

Sets quickly

A

Agarose

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46
Q

Can be refrigerated

A

Agarose

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47
Q

Non toxic

A

Agarose

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48
Q

Pore size can be manipulated

A

Agarose

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49
Q

Buffers can alter the resolution and run times

A

Agarose

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50
Q

Large range of DNA can be separated

A

Agarose

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51
Q

Produce reproducible and consistent results

A

Polyacrylamide

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52
Q

Pores uniform in size (smaller)

A

Polyacrylamide

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53
Q

Non-reactive/inert with samples

A

Polyacrylamide

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54
Q

Very high resolving power -clear DNA fragments

A

Polyacrylamide

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55
Q

Pore size can be manipulated

A

Polyacrylamide

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56
Q

Buffers can alter the resolution and run times

A

Polyacrylamide

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57
Q

Do not have a uniform pore size

A

Agarose

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58
Q

Low percentage gels: may be weak and may break

A

Agarose

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59
Q

High percentage gels: brittle and may not set evenly

A

Agarose

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60
Q

Potent neurotoxin (Acrylamide)

A

Polyacrylamide

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61
Q

PPE must be used though

A

Polyacrylamide

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62
Q

PA is non-toxic

A

Polyacrylamide

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63
Q

Bubble may form in pouring -due to chem rx

A

Polyacrylamide

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64
Q

Longer wait time

A

Polyacrylamide

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65
Q

Based on the length of plasmid/bp ; ↓agarose conc = weaker ; Closest to the lower limit

A

Agarose Gel Conc (1-2%)

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66
Q

: Large pores size; may be weak and may break

A

Low percentage gels

67
Q

: Small pore size; brittle and may not set evenly

A

High percentage gels

68
Q
  • the larger fragments are much better resolved in the [?] gel
A

0.7%

69
Q
  • the small fragments separated best in [?] agarose (better separation)
A

1.5%

70
Q
  • the [?] fragment i indicated in each lane
A

1000 bp

71
Q

are sufficiently pure to avoid problems such as electroendosmosis

A
  • Agarose preparations
72
Q

a solvent flow toward one of the electrodes, usually the cathode (negative), in opposition to the DNA or RNA migration,

A

electroendosmosis

73
Q

slows and distorts the migration of the samples, reducing resolution and smearing the band

A

electroendosmosis

74
Q

(prevented by choosing the correct agarose conc)

A

electroendosmosis

75
Q

QUESTION: You wish to perform an electrophoretic resolution of your restriction enzyme-digested DNA. The sizes of the expected fragments range from 100 to 500 bp. You discover two agarose gels polymerizing on the bench. One is 0.5% agarose; the other is 2% agarose. Which one might you use to resolve your fragments?

A

2%: used for shorter DNA fragements

76
Q

Solution that will conduct electricity because of their hydrogen ion concentration and maintain the pH

A

Buffers

77
Q

Gives the charge on the agarose gel to allow flow of elec to the gel matrix

A

Buffers

78
Q

: chelator of nucleases

A

EDTA

79
Q

cancels out enzymes that coud disrupt DNA against DNAses

A

EDTA

80
Q

Low buffering capacity

A

tris-acetate-EDTA (TAE)

81
Q

High buffering capacity (does not change the pH)

A

tris-borate-EDTA (TBE)

82
Q

Fast DNA migration (better conductivity)

A

tris-acetate-EDTA (TAE)

83
Q

Slow DNA migration

A

tris-borate-EDTA (TBE)

84
Q

Overheats and exhausted during extended/ repeated electrophoresis

A

tris-acetate-EDTA (TAE)

85
Q

Adv: Won’t overheat quickly during long runs (controls temp to prevent melting)

A

tris-borate-EDTA (TBE)

86
Q

Good for large fragments (>2kb)

A

tris-acetate-EDTA (TAE)

87
Q

For smaller fragments (<2kb)

A

tris-borate-EDTA (TBE)

88
Q

Non toxic

A

tris-acetate-EDTA (TAE)

89
Q

Carcinogenic

A

tris-borate-EDTA (TBE)

90
Q

Absent Borate

A

tris-acetate-EDTA (TAE)

91
Q

Borate present as enzyme inhibitor (more expensive)

A

tris-borate-EDTA (TBE)

92
Q

Lower cost

A

tris-acetate-EDTA (TAE)

93
Q

Higher cost

A

tris-borate-EDTA (TBE)

94
Q

used at 1X for the electrophoresis of RNA as the running buffer to separate RNA samples on agarose and formaldehyde-agarose (denaturing) gels

A

MOPS buffer (Buffer additive)

95
Q

Adds mass/ density to the DNA sample so that it will go into the well makes it sink to the bottom

A

Loading dye

96
Q

mixed w/ DNA samples and added thru the wells created from the gel comb

A

Loading dye

97
Q

absence: DNA could float off the gel

A

Loading dye

98
Q

(sinkers: [?])

A

Glycerol, sucrose and ficoll

99
Q

Adds blue color so you can see what you are pipetting

A

Tracking dye

100
Q

Tracking dyes:

A

(Bromophenol blue – most common, Cresol Red, Orange G, tartrazine, xylene cyanol)

101
Q

to know whether electrophoresis (EP) has started; EB can be added before starting EP

A

Tracking dye

102
Q

Contains DNA fragments with known sizes that are compared to our experimental setups and are used to estimate the sizes of DNA fragments in your DNA sample

A

Standard/Molecular weight markers/DNA ladder

103
Q

basis to compare the DNA set-up

A

Standard/Molecular weight markers/DNA ladder

104
Q

should be performed on the same agarose gel, not separated to prevent varying temp, run, conc

A

DNA ladder

105
Q
  1. What lane contains the longest DNA fragment?
A

1

106
Q
  1. What lane contains the shortest DNA fragment?
A

2

107
Q
  1. What lane contains 1500bp DNA fragment?
A

3

108
Q

RNA ladders are usually provided with a loading buffer containing a

A

denaturant/ denaturing agents.

109
Q

: for single-stranded DNA/linear RNA

A

Denaturing agents

110
Q

Denaturing agents

A

 Formide
 Urea/Chaotropic agents
 Sodium dodecyl sulphate/SDS (thermofisher)

111
Q

Denaturants purpose:
1. help maintain RNA in [?] (linear in shape)
2. Prevents the formation of [?] (folding)
3. Allow more predictable sample [?] results.

A

single-stranded form

secondary structure

migration and separation

112
Q

When performing RNA gel electrophoresis, [?] should be avoided, because their use under denaturing conditions can lead to atypical separation patterns due to separation of the double strands.

A

DNA ladders

113
Q

run under the condition that disrupts the natural/native structure of DNA/RNA or protein, which are unfolded into linear chains.

A

Denaturing gels

114
Q

run under conditions that no disruption of structure is introduced to analytes.

A

Non-denaturing (native) gel

115
Q

The speed of these macromolecules moving through a gel depends only on their linear length and the mass-to-charge ratio; thus, only the primary structure is analyzed.

A

Denaturing gels

116
Q

In this case, the cross-sectional area of the macromolecule, in addition to the molecular mass and intrinsic charge, is also a factor for gel separation, allowing for analysis of all four levels (primary, secondary, tertiary, and quaternary levels) of the biomolecular structure.

A

Non-denaturing (native) gel

117
Q

Urea is usually to denature DNA or RNA, while sodium dodecyl sulfate is used for protein denaturing

A

Denaturing gels

118
Q

Fluorescent dye

A

Ethidium Bromide

119
Q

May be added during the making of agarose gel or after the migration

A

Ethidium Bromide

120
Q

DNAs are visualized by staining the gel

A

Ethidium Bromide

121
Q

will bind to DNA and will fluoresce in UV light.

A

Ethidium Bromide

122
Q

Carcinogenic

A

Ethidium Bromide

123
Q

: UV lightbox to visualize the EtBr stained gel

A

Transilluminator

124
Q

Safer alternative, more expensive

A

SYBR Green/ SYBR Safe/SYBR Gold

125
Q

seen under UV

A

SYBR Green/ SYBR Safe/SYBR Gold

126
Q

ELECTROPHORESIS PROCEDURE

A
127
Q
  1. Prepare agarose
     [?] o Ex: 0.5 agar in 50mL buffer will result to 1%
     Once the tablets have dissolved chemically by stirring, heat the solution in a microwave at full power for short bursts of [?] (prevent over boiling; as soon as bubbles appear)
     Overboiling: [?] (buffer as a diluent)
     Stop when you see [?]
     Add [?
     [?] of DNA Safe Stains to every [?] of gel solution
     [?]: It emits green fluorescence when bound to dsDNA (double) and red fluorescence when bound to ssDNA (single) or RNA
A

Gel percentage=(grams of agarose / milliliters of buffer) × 100%

20-30 seconds

Increased percentage concentration and high ionic concentration

bubbles

ethidium bromide or DNA safe stain

1 uL ; 20 mL

DNA safe stains

128
Q
  1. Preparing the gel box for casting
     Ensure the [?] are installed correctly, then install one of the [?].
     Place the [?]
A

black buffer dams ; combs

tape

129
Q
  1. Pouring the gel
     Once the agarose is fully melted and dissolved, let the solution cool to about [?]. The beaker should feel hot, but not too hot to touch.
     [?]: warp/melt the casting tray and comb
     Pour at [?] mark on a level surface (or 0.5 cm/5 mm)
     It will take [?] to solidify at RT, one can place it into the fridge
A

55°C

Too hot

6-7mm

30 minutes

130
Q

affects the DNA fragments

A

Thickness of the gel

131
Q
  1. Remove the comb and buffer dams/ tape
    Be careful not to damage the [?] as you remove the comb and shutters. When removing the [?], make sure not to pierce the gel.
A

gel

comb

132
Q
  1. Gel buffer
    Use your prepared 0.5x TBE buffer solution again, and pour it over the gel until the gel is fully covered. The buffer should reach about [?] above the gel.
A

2-3mm

133
Q
  1. Loading Dye
    Before loading each sample, you need to add [?] to each sample
A

loading dye

134
Q
  1. Loading the gel with sample
     Using a fresh pipette tip, load [?] (w/ DNA sample and loading dye) of the sample into an empty well. Discard your pipette tip, then make a note so that you remember which sample is in which well.
     Repeat this for each sample, taking care to use a [?] each time.
A

5ul

fresh pipette tip

135
Q
  1. Run gel at constant voltage until band separation occurs
     Gently close the [?] by sliding the lid onto the gel tray. Then connect the leads to the power supply. Be careful not to spill anything.
     Longer electrophoretic runs will increase the [?] between fragments
     Adequate separation is important for analysis of [?], especially those that are close in size.
     However, if the electrophoresis is conducted for too long, DNA bands may [?] of the gel
     The [?], the faster the DNA will travel through the gel. However, voltages that are too high can possibly [?] of DNA bands.
     The [?] affect electrophoretic separation. For example, DNA samples will migrate faster in a 0.8% gel compared to a 1% gel. Likewise, samples will migrate faster in a 20 ml gel (6 mm thick) versus a 30 ml gel (8 mm thick) with the same 7 x 7 centimeter dimensions.
A

gel box

separation

DNA fragments

migrate off the end

higher the voltage ; melt the gel or cause smearing or distortion

gel concentration and volume (thickness)

136
Q

depends on the bp being tested

A

Voltage

137
Q
  1. View DNA on UV light box and document results
     Identify the DNA base pair based on the
A

DNA ladder

138
Q
  1. DNA/RNA purification from a mixture
     Separate the DNAs in [?]
     Cut the required [?]
     Extract the DNA from gel thru [?]
A

Agarose gel

(DNA + Gel) band

elution or other methods

139
Q

 Requires training and precautions for radioactive safety

A

Radioactive stains

140
Q

 Detects or labels samples by radioactive probes or nucleotides

A

Radioactive stains

141
Q

 Common for detection of oligonucleotides

A

Radioactive stains

142
Q

 Considered most sensitive (but poses health risk)

A

Radioactive stains

143
Q

Radioactive stains labels

A

32P; 33P; 3H labels

144
Q

 the gel is exposed to X-ray film after ectrophoresis for documentation

A

Autoradiography

145
Q

 The intensities of radiolabeled bands may be measured by densitometry for quantitation.

A

Autoradiography

146
Q

Plasmid which is

A

4070bp (4kb)

147
Q

: DNA cutting enzyme which recognize or a few target sequences and cuts DNA a– those sequences

A

Ecor1

148
Q

 acts as a cutting enzyme/scissor

A

Ecor1

149
Q

 restriction enzyme found in E. coli

A

Ecor1

150
Q

 seen using eletrophoresis

A

Ecor1

151
Q
  1. [?] cleave DNA into smaller segments of various sizes.
  2. [?]are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes.
  3. When an electric current is passed through the chamber, DNA fragments move toward the [?].
  4. Smaller DNA segments move [?] than larger DNA segments.
A

Restriction enzymes

DNA segments

positively-charged cathode

faster and farther

152
Q

Cut and combine to a gene with [?] with a pasting enzyme (DNA ligase) to create a new plasmid

A

1,000 bp

153
Q

Get the 1,200 bp and pair with a gene with [?] to create another recombinant DNA

A

2,870 bp

154
Q

Agarose tablets/powder are placed in the beaker

A
  1. Prepare agarose
155
Q

Buffer/diluent: tris-borate EDTA or distilled water

A
  1. Prepare agarose
156
Q

2.5 uL DNA stain : 50 mL of gel solution

A
  1. Prepare agarose
157
Q

Comb serves as a mold for the wells

A
  1. Preparing the gel box for casting
158
Q

Buffer dams/tape: black

A
  1. Preparing the gel box for casting
159
Q
  1. Remove the comb and buffer dams/ tape
A

Wells are created

160
Q

Add/overlay the agarose gel w/ the gel buffer

A
  1. Gel buffer
161
Q

Pipette loading dyes to the sample (sinker to have higher density: glycerol, stain: bromophenol blue)

A
  1. Loading Dye
162
Q

Connect the lids and set at 30 mins, 50 volts
o 1 hr: DNA fragments migrate further

A
  1. Run gel at constant voltage until band separation occurs
163
Q

Cap the gel tray to prevent electrocution
-Loaded with loading dye

A
  1. Run gel at constant voltage until band separation occurs
164
Q

Clue to prevent overpassing of agarose gel:

A

migration is 3.5 – 4.0 cm (close to 60% of the agarose gel or 75%)

[Molecular Biology] Lec (13 decks)

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