Quiz 2: Lab Agarose Gel Electrophoresis Flashcards
What is Agarose gel electrophoresis the most effective way of?
Separating DNA fragments of varying sizes ranging from 100 bp to 25 kb
What happens to agarose polymers during gelation?
During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel’s molecular sieving properties
What did the use of agarose gel revolutionize?
Separation of DNA
What is done to separate DNA using agarose gel electrophoresis?
DNA is loaded into pre-cast wells in the gel and a current applied
The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode
Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight
What is the leading model for DNA movement through agarose gel?
“Biased Reptation” whereby the leading edge moves forward and pulls the rest of the molecule along
The rate of migration of a DNA molecule through a gel is determined by the following: (7)
1) size of DNA molecule
2) agarose concentration
3) DNA conformation
4) voltage applied
5) presence of ethidium bromide
6) type of agarose
7) electrophoresis buffer
How can the DNA molecules be visualized after separation?
Under UV light after staining with an appropriate dye
By following this protocol, students should be able to: (8)
- Understand the mechanism by which DNA fragments are separated within a gel matrix
- Understand how conformation of the DNA molecule will determine its mobility through a gel matrix
- Identify an agarose solution of appropriate concentration for their needs
- Prepare an agarose gel for electrophoresis of DNA samples
- Set up the gel electrophoresis apparatus and power supply
- Select an appropriate voltage for the separation of DNA fragments
- Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands
- Determine the sizes of separated DNA fragments
What does loading dye help to do?
Track how far your DNA sample has traveled, and allows the sample to sink into the gel
What do you do with the running buffer?
Add enough running buffer to cover the surface of the gel
Important to use the same running buffer as the one used to prepare the gel
What should always be loaded along with the experimental samples?
Appropriate DNA size marker (DNA standard/ DNA Ladder)
What are the cathode and anode?
Cathode (Black leads) should be closer to the wells than the anode
Anode (Red leads)
Agarose gel electrophoresis has proven to be an efficient and effective way of what?
Separating nucleic acids
What is molecular sieving determined by?
Size of pores generated by the bundles of agarose in the gel matrix
The higher the concentration of agarose,
The smaller the pore size
What is EtBr?
Most common reagent used to stain DNA in agarose gels
How does EtBr work?
What does it allow for?
EtBr works by intercalating itself in the DNA molecule in a concentration dependent manner
This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity
(Because of its positive charge, the use of EtBr is reduces DNA migration by 15%)
What stain did we use for DNA in agarose gel?
Methyl blue?
What are the 3 major purposes of loading dyes used in gel electrophoresis?
Add density to the sample, allowing it to sink into the gel
Dyes provide color and simplify the loading process
Dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated
How can the exact sizes of separated DNA fragments be determined?
By plotting the lot of the molecular weight for the different bands of a DNA standard against the distance traveled by each band
What does the DNA standard (ladder) contain?
Mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples
What is important to note?
What moves through the gel the fastest?
What moves the slowest?
Different forms of DNA move through the gel at different rates
Supercooled plasmid DNA, because of its compact formation
Followed by a linear DNA fragment of the same size
Open circular form traveling the slowest
What are the most common gel running buffers?
TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA)
