LESSON 9 GEL ELECTROPHORESIS Flashcards
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
gel electrophoresis?
A technique used to separate DNA fragments according to their size.
gel electrophoresis?
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
- PCR test
- DNA cloning/ Recombinant technology
Sample has already run thru the electric current and have migrated from the
negatively charged(cathode) to the positively charged (anode) area
: diabetic patients (9 patients)
TGF-B gene
o standard/reagent/DNA ladder
M (molecular weight markers)
o serves as a basis for the length of DNA with a unit of “bp” (base pair)
M (molecular weight markers)
= 1 kbp
1,000 bp
: DNA ladder
Lane 1
: has a DNA fragment of 1,500 bp
Lane 2
: has a DNA fragment of 600 bp (with TGF-B gene)
Lane 3-9
: fluorescent dye stain
Ethidium bromide
Used for visualization under UV light/transilluminator
Ethidium bromide
To get the fragment (?), elute or cut the agarose gel (DNA portion) → wash
600 bp
acts as a strainer for the separation of particles
gel in electrophoresis
: fastest to migrate
Small DNA/RNA fragemnts/shorter base pairs
voltages involved
(positive and negative charges)
separation according to
size of bp
2 kbp from 10 bp =
2 kbp gene
DNA fragments are [?] (loaded at the cathode), so they move towards the positive electrode (anode).
negatively charged
presence of phosphate group (phosphoric acid)
negatively charged
Separation takes
30-45 mins
: DNA fragments will overpass
Long electrical runs
Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel [?] than large ones.
faster
Voltage:
Depends on the protocol
60-120 V
Separated in [?]; test is optimized; proper migration to properly identify their sizes
bands
REAGENTS EQUIPMENTS AND REAGENTS/ELECTROPHORESIS SYSTEM
Power supply and chamber
Gel casting tray/gel box/ gel chamber:
Gel comb:
Buffer dam or tapes:
a source of power supply
Power supply and chamber
: hold/mold the agarose
Gel casting tray/gel box/ gel chamber
: create holes or sample wells in the gel where the samples are loaded
Gel comb
: limits the movement of the agarose gel
Buffer dam or tapes
Matrix where samples are loaded and DNA migrates
Agarose
Commonly used in gel electrophoresis
Agarose
For big DNA fragments from 50-20,000bp
Agarose
High resolving power for short 5-500bp
Polyacrylamide
Horizontal
Agarose
Vertical
Polyacrylamide
High agarose conc: smaller pore size
Agarose
Routinely 6-15%
Polyacrylamide
Set as it cools
Agarose
Sets by chemical reaction once crosslinking occurs
Polyacrylamide
Polysaccharide extracted from sea weed
Agarose
Crosslinked polymer of acrylamide
-Neurotoxin
-P.A.: nontoxic
Polyacrylamide
Easy to prepare
Agarose
Sets quickly
Agarose
Can be refrigerated
Agarose
Non toxic
Agarose
Pore size can be manipulated
Agarose
Buffers can alter the resolution and run times
Agarose
Large range of DNA can be separated
Agarose
Produce reproducible and consistent results
Polyacrylamide
Pores uniform in size (smaller)
Polyacrylamide
Non-reactive/inert with samples
Polyacrylamide
Very high resolving power -clear DNA fragments
Polyacrylamide
Pore size can be manipulated
Polyacrylamide
Buffers can alter the resolution and run times
Polyacrylamide
Do not have a uniform pore size
Agarose
Low percentage gels: may be weak and may break
Agarose
High percentage gels: brittle and may not set evenly
Agarose
Potent neurotoxin (Acrylamide)
Polyacrylamide
PPE must be used though
Polyacrylamide
PA is non-toxic
Polyacrylamide
Bubble may form in pouring -due to chem rx
Polyacrylamide
Longer wait time
Polyacrylamide
Based on the length of plasmid/bp ; ↓agarose conc = weaker ; Closest to the lower limit
Agarose Gel Conc (1-2%)
: Large pores size; may be weak and may break
Low percentage gels
: Small pore size; brittle and may not set evenly
High percentage gels
- the larger fragments are much better resolved in the [?] gel
0.7%
- the small fragments separated best in [?] agarose (better separation)
1.5%
- the [?] fragment i indicated in each lane
1000 bp
are sufficiently pure to avoid problems such as electroendosmosis
- Agarose preparations
a solvent flow toward one of the electrodes, usually the cathode (negative), in opposition to the DNA or RNA migration,
electroendosmosis
slows and distorts the migration of the samples, reducing resolution and smearing the band
electroendosmosis
(prevented by choosing the correct agarose conc)
electroendosmosis
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?
2%: used for shorter DNA fragements
Solution that will conduct electricity because of their hydrogen ion concentration and maintain the pH
Buffers
Gives the charge on the agarose gel to allow flow of elec to the gel matrix
Buffers
: chelator of nucleases
EDTA
cancels out enzymes that coud disrupt DNA against DNAses
EDTA
Low buffering capacity
tris-acetate-EDTA (TAE)
High buffering capacity (does not change the pH)
tris-borate-EDTA (TBE)
Fast DNA migration (better conductivity)
tris-acetate-EDTA (TAE)
Slow DNA migration
tris-borate-EDTA (TBE)
Overheats and exhausted during extended/ repeated electrophoresis
tris-acetate-EDTA (TAE)
Adv: Won’t overheat quickly during long runs (controls temp to prevent melting)
tris-borate-EDTA (TBE)
Good for large fragments (>2kb)
tris-acetate-EDTA (TAE)
For smaller fragments (<2kb)
tris-borate-EDTA (TBE)
Non toxic
tris-acetate-EDTA (TAE)
Carcinogenic
tris-borate-EDTA (TBE)
Absent Borate
tris-acetate-EDTA (TAE)
Borate present as enzyme inhibitor (more expensive)
tris-borate-EDTA (TBE)
Lower cost
tris-acetate-EDTA (TAE)
Higher cost
tris-borate-EDTA (TBE)
used at 1X for the electrophoresis of RNA as the running buffer to separate RNA samples on agarose and formaldehyde-agarose (denaturing) gels
MOPS buffer (Buffer additive)
Adds mass/ density to the DNA sample so that it will go into the well makes it sink to the bottom
Loading dye
mixed w/ DNA samples and added thru the wells created from the gel comb
Loading dye
absence: DNA could float off the gel
Loading dye
(sinkers: [?])
Glycerol, sucrose and ficoll
Adds blue color so you can see what you are pipetting
Tracking dye
Tracking dyes:
(Bromophenol blue – most common, Cresol Red, Orange G, tartrazine, xylene cyanol)
to know whether electrophoresis (EP) has started; EB can be added before starting EP
Tracking dye
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
Standard/Molecular weight markers/DNA ladder
basis to compare the DNA set-up
Standard/Molecular weight markers/DNA ladder
should be performed on the same agarose gel, not separated to prevent varying temp, run, conc
DNA ladder
- What lane contains the longest DNA fragment?
1
- What lane contains the shortest DNA fragment?
2
- What lane contains 1500bp DNA fragment?
3
RNA ladders are usually provided with a loading buffer containing a
denaturant/ denaturing agents.
: for single-stranded DNA/linear RNA
Denaturing agents
Denaturing agents
Formide
Urea/Chaotropic agents
Sodium dodecyl sulphate/SDS (thermofisher)
Denaturants purpose:
1. help maintain RNA in [?] (linear in shape)
2. Prevents the formation of [?] (folding)
3. Allow more predictable sample [?] results.
single-stranded form
secondary structure
migration and separation
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.
DNA ladders
run under the condition that disrupts the natural/native structure of DNA/RNA or protein, which are unfolded into linear chains.
Denaturing gels
run under conditions that no disruption of structure is introduced to analytes.
Non-denaturing (native) gel
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.
Denaturing gels
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.
Non-denaturing (native) gel
Urea is usually to denature DNA or RNA, while sodium dodecyl sulfate is used for protein denaturing
Denaturing gels
Fluorescent dye
Ethidium Bromide
May be added during the making of agarose gel or after the migration
Ethidium Bromide
DNAs are visualized by staining the gel
Ethidium Bromide
will bind to DNA and will fluoresce in UV light.
Ethidium Bromide
Carcinogenic
Ethidium Bromide
: UV lightbox to visualize the EtBr stained gel
Transilluminator
Safer alternative, more expensive
SYBR Green/ SYBR Safe/SYBR Gold
seen under UV
SYBR Green/ SYBR Safe/SYBR Gold
ELECTROPHORESIS PROCEDURE
- 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
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
- Preparing the gel box for casting
Ensure the [?] are installed correctly, then install one of the [?].
Place the [?]
black buffer dams ; combs
tape
- 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
55°C
Too hot
6-7mm
30 minutes
affects the DNA fragments
Thickness of the gel
- 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.
gel
comb
- 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.
2-3mm
- Loading Dye
Before loading each sample, you need to add [?] to each sample
loading dye
- 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.
5ul
fresh pipette tip
- 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.
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)
depends on the bp being tested
Voltage
- View DNA on UV light box and document results
Identify the DNA base pair based on the
DNA ladder
- DNA/RNA purification from a mixture
Separate the DNAs in [?]
Cut the required [?]
Extract the DNA from gel thru [?]
Agarose gel
(DNA + Gel) band
elution or other methods
Requires training and precautions for radioactive safety
Radioactive stains
Detects or labels samples by radioactive probes or nucleotides
Radioactive stains
Common for detection of oligonucleotides
Radioactive stains
Considered most sensitive (but poses health risk)
Radioactive stains
Radioactive stains labels
32P; 33P; 3H labels
the gel is exposed to X-ray film after ectrophoresis for documentation
Autoradiography
The intensities of radiolabeled bands may be measured by densitometry for quantitation.
Autoradiography
Plasmid which is
4070bp (4kb)
: DNA cutting enzyme which recognize or a few target sequences and cuts DNA a– those sequences
Ecor1
acts as a cutting enzyme/scissor
Ecor1
restriction enzyme found in E. coli
Ecor1
seen using eletrophoresis
Ecor1
- [?] cleave DNA into smaller segments of various sizes.
- [?]are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes.
- When an electric current is passed through the chamber, DNA fragments move toward the [?].
- Smaller DNA segments move [?] than larger DNA segments.
Restriction enzymes
DNA segments
positively-charged cathode
faster and farther
Cut and combine to a gene with [?] with a pasting enzyme (DNA ligase) to create a new plasmid
1,000 bp
Get the 1,200 bp and pair with a gene with [?] to create another recombinant DNA
2,870 bp
Agarose tablets/powder are placed in the beaker
- Prepare agarose
Buffer/diluent: tris-borate EDTA or distilled water
- Prepare agarose
2.5 uL DNA stain : 50 mL of gel solution
- Prepare agarose
Comb serves as a mold for the wells
- Preparing the gel box for casting
Buffer dams/tape: black
- Preparing the gel box for casting
- Remove the comb and buffer dams/ tape
Wells are created
Add/overlay the agarose gel w/ the gel buffer
- Gel buffer
Pipette loading dyes to the sample (sinker to have higher density: glycerol, stain: bromophenol blue)
- Loading Dye
Connect the lids and set at 30 mins, 50 volts
o 1 hr: DNA fragments migrate further
- Run gel at constant voltage until band separation occurs
Cap the gel tray to prevent electrocution
-Loaded with loading dye
- Run gel at constant voltage until band separation occurs
Clue to prevent overpassing of agarose gel:
migration is 3.5 – 4.0 cm (close to 60% of the agarose gel or 75%)
