Lecture Four - Restriction Mapping/Electrophoresis
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What is Electrophoresis?
Motion of charged particles in an External Electric Field
What is the charge of DNA?
Which is correct (for tagging electrophoresis gel)?
What is Ethidium Bromide?
An intercalating agent used in Labelling DNA fragments on electrophoresis gel.q
What was used before Ethidium Bromide to label Electrophoresis?
The DNA was radioactively labelled and had to be assayed.
Why did Ethidium Bromide replace Radiocactive Assay?
Radioctive Assays are slow and cumbersome.
How does Ethidium Bromide work?
It binds non-specifically to the DNA and fluoresces under UV light.
What are the two types of Gel?
What gel is preferred for DNA electrophoresis?
How do the Gels work?
They form sieve-like pores that Retard molecules by size.
Why is Polyacrylamide better at seperating smaller molcules?
It has smaller pores
What are some advantages of Polycarylamide gel?
- Better Resolving Power
- Better at Seperating Smaller Molecules
What are some Disadvantages of Polyacrylamide?
- Difficult to prepare (polymerize)
- MonoAcrylamide is considered a Neurotoxin
What are some Advantages of Agarose?
- Horizontal Configuration
Which Gel has the greatest effective range of DNA sizes?
How does p.d affect DNA migration through a gel?
- At low voltages Migration is proportional to potential difference.
- At high voltages motion is more (exponential?).
What is the most effective voltage?
How can you alter the pore sizes in Agarose Gel?
Change the Agarose concentration
Why might you choose to use smaller pores?
For smaller DNA fragments
How does pore size affect range?
Smaller pores -> Less Range of suitable sizes
What is the maximum size of DNA that can be used in Electrophoresis?
- 30kb. DNA larger than this does not seperate.
- Use Sequencing
Uncut plasmids usually show three bands on an electrophoresis. What do they represent?
- In order of least migration:
- 1. Linear DNA
- 2. Nicked Plasmids
- 3. Supercoiled Plasmids
What is a Doublet with respect to Gel Electrophoresis?
Two bands very close together
Sometimes plasmids show one distinct bands and a doublet. What two bands make the doublet?
- The Linear DNA
- The Nicked DNA
Three bands are seen from a single type of unmodfied plasmid DNA. Why does this occur?
Usually some damage will occur during extraction resulting in Nicking or Complete breaking of the plasmid.
What is a Nicked plasmid?
A plasmid with a single stand of its ds-DNA broken which causes it to relax (from it's supercoiled form) into a more circular shape.
What protein is responsible for Supercoiling DNA?
Which conformation of plasmid migrates fastest?
Which conformation of plasmid migrates slowest?
What effect does Ethidium Bromide have on DNA Migration?
It slows it down
How does Ethidum Bromide affect the charge of DNA?
It binds and reduces the negative charge of the DNA molecule.
How does Ethidium Bromide affect the structure of DNA?
Being a bulky molecule it alters DNA's stiffness and strength.
What three features of DNA does Ethidum Bromide affect?
- It's charge
- It's Stiffness
- It's strength
What is the solidifying temperature of Agarose?
What is the Melting Temperature of Agarose?
What is Low Melting Point Agarose?
Agarose with a much lower melting point than normal.
What is the Melting Point of Low Melting Point Agarose?
Why might Low Melting Point Agarose be useful?
- Altering DNA without extraction (melt and modify)
- Bacterial Transformation can occur in the gel
- Low Melting Point Agarose can stabillize DNA
Why might it be essential to minimize purifications?
Because purification always results in some loss of DNA
What happens if no buffer is present in Gel Electrophoresis?
No migration will occur due to insufficient ionic strength
What happens if a buffer has too great an ionic strength?
The gel could overheat
What is the basic setup of an Agarose gel with regards to the buffer?
- First, agarose is dissolved in a buffer
- The gel is then solidified and placed in a buffer chamber
- The gel is now ready for loading
What is the purpose of the loading dye?
- To see if the well is punctured
- Dissolved sucrose increases density meaning DNA sinks to the bottom
- Move predictably in an electric field (like a rough placemarker for DNA)
What are some considerations when choosing a loading dye?
- Size of DNA
- Potential Obscuring
How does Ethidium Bromide work as a DNA label?
- It binds nonspecifically to the DNA and absorbs UV light.
- The UV light is then re-emitted as red-orange light
What is the effect of binding DNA on the absorbtion and emission of photons from Ethidium Bromide?
It makes it more fluorescent
What are some disadvantages of Ethidium Bromide?
- It is susceptible to contamination
- Gloves must be warn (mutagenic)
- Best added after gel is run
What is Sybr Gold?
An intercalating agent used in detecting DNA for electrophoresis
What are some disadvantages of Sybr Gold?
- It is expensive
- It is packaged with DMSO which will carry it across skin
What are some pros of Sybr Gold?
- It binds to DNA
- It is non-mutagenic
Why might anything that binds DNA be potentially dangerous?
Because it interacts directly, it may cause mutations or damage.
How do you determine the size of DNA on an Agarose Gel?
A standard curve produced by some standard form(s) of DNA
What are some Standard DNAs used?
- Known DNA sequences digested by known enzymes (e.g Lambda + HindIII)
- Commercial Preparations (2-log, 1kb plus etc)
Can a standard curve be reused?
No, a new curve must be made for every gel
Where should you measure your bands from?
Usually the leading edge, but it doesn't matter so long as you are consistent
How many points of a standard curve should you plot?
What is Pulsed Field Gel Electrophoresis?
Electrophesis using an alternating electric field
What is Agarose made from?
HeteroPolymers consisting of two types of Galactose molecules
What are the two types of Galactose that make one agarose monomer?
- 3,6-anyhdro L-Galactose
What are some factors influencing DNA migraion through electrophoresis gel?
- Electrical Current/Voltage
- DNA Size
- Concentration of Agarose
- DNA Conformation
- Ethidium Bromide
- Agarose Type
- Buffer used
How can you measure the rate of migration of a known size of DNA?
Take the recirocal of log the log of the base pairs (1/(log10(bp))
If you wanted to perform Gel Electrophoresis on DNA that is 15kb, what concentration of Agarose might you use?
If you wanted to perform Gel Electrophoresis on DNA that is 5kb, what concentration of Agarose might you use?
If you wanted to perform Gel Electrophoresis on DNA that is 60bp, what concentration of Agarose might you use?
How is Low Melting Point Agarose different from regular Agarose?
It has been modified by Hyroxyethylation
What are the two key purposes of an Electrophoresis buffer?
- Provide ions to carry current
- Maintenance of pH
What are the two most common buffers in Gel Electrophoresis?
- Tris-Acetate + EDTA (pH8.0)
- Tris-Borate + EDTA (pH8.0)
How are buffer solutions stored?
At high concentrations
What is the working concentration of TAE and TBE?
0.5x - 1.0x
What is TAE?
Tris-Acetate and EDTA, it is an electrophoresis buffer.
What is TBE?
Tris-Borate & EDTA, it is an Electrophoresis buffer
Which of the two main Buffers has the highest buffering capacity?
Of the two buffers, which gives faster migration of linear dsDNA?
What buffer might I use for large DNA molecule (High Molecular Weight)?
What buffer might I use for Small DNA molecule (Low Molecular Weight)?
Which buffer is best for mixtures of DNA?
What are some advantages of TBE?
- High Buffering Capacity
Which of TBE or TAE is more expensive?
What is the minimum amount of DNA Ethidium Bromide can detect?
What is the stock concentration f Ethidium Bromide?
What is the practical concentration of Ethidium Bromide. When should it be added?
0.5ug/mL after dissolving Agarose
What is a problem encountered with Electrophoresis of RNA?
Complex Secondary strctures can give strange migrational patters (i.e it misbehaves)
How are the problems with RNA Gel electrophoresis overcome?
- Using Agarose Gels Containing Formaldehyde
- (Or) Urea Containing Polyacrylamide Gels
How might you go about Pulsed Field Gel Electrophoresis?
- Immobillize Cells in Low Melting Point Agarose
- Lyse cells
- Digest DNA with a Rare Cutter
Why is Low Melting Point Agarose Gel used to Immobillize the cells?
It can be melted and the cells whithin further modifed
What is used to lyse the cells?
- Proteinase K
What rare cutter is used in the Digestion of Cells in PFGE?
Often NotI (8bp recognition sequence)
What is the basic layout of a PFGE setup?
The gel (and buffer) surrounded by lots of evenly spaces electrodes (often in a hexagonal arrangement)
What is a consequence of not spacing electrodes regularly?
How do PFGE work?
- The alternating field causes the DNA to distort and snake through the pores of the gel reorienting with every "pulse"
- Larger molecules take longer to reorient
How might you set up a Formaldehyde Gel for RNA?
- Heat the RNA in it's buffer then add Formamide and Ethidium Bromide
- Next Make a solution of Agarose and Formaldehyde
What should the concentration of Agarose be in a formalyde gel?
What should the concentration of formaldehyde be in a Formalyhyde Gel for RNA?
What is an Advantage of Polycrylamide Gel for RNA over Agarose?
Better resolution (diference of 1nt)
How is Acrylamide Polymerized?
- The reaction is Initiated by free radicals initiated by Ammonium Persulfate.
- Catalysed by TEMED
What are the two key steps in Restriction Mapping?
How is separation achieved in Restriction Mapping?
What are some uses of Restriction Mapping?
- Determining Information about a Clone
- Determining orientation of DNA insterted into a plasmid
How is Restriction Mapping Useful in Systematics?
Looking at Relatedness
What informaion about a clone can Restriction Mapping give you?
- Locating a particular fragment
- Size Determination
- Locating restriction sites for future reference
Why might Restriction Mapping be better than Sequencing?
- Sequencing is limited to 600bp
- A starting point and comlimentary nucleotides must be added
- It takes time
What is the length of an average Prokaryote Gene?
What is the length of an Average Eukaryote gene?
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