Download - Mdb lab 2 lecture agarose gels
EXPERIMENTAL OVERVIEW
PCR a DNA fragment using a plasmid template
Identify and purify the PCR product
Digest the product with suitable restriction enzymes
Ligate the product into a suitably digested cloning vector
Transform E. coli
Identify colonies containing the insert
Confirm the clone by miniprep of DNA and restriction digest
Perform large-scale DNA preparation
Sequence the chosen subclone
Analyze sequence by database searching/bioinformatics
EXPERIMENTAL OVERVIEW
PCR a DNA fragment using a plasmid template
Identify and purify the PCR product
Digest the product with suitable restriction enzymes
Ligate the product into a suitably digested cloning vector
Transform E. coli
Identify colonies containing the insert
Confirm the clone by miniprep of DNA and restriction digest
Perform large-scale DNA preparation
Sequence the chosen subclone
Analyze sequence by database searching/bioinformatics
Agarose Gel Electrophoresis
--Method for separating DNA fragments by size (also topology) --Often used (with DNA size standards) to determine the size of DNA fragments.
Agarose Gel Electrophoresis
--Method for separating DNA fragments by size (also topology) --Often used (with DNA size standards) to determine the size of DNA fragments. 1. Linear DNA fragments migrate through an agarose gel matrix with a mobility that is inversely
proportional to the log10 of their molecular weight.
Agarose Gel Electrophoresis
--Method for separating DNA fragments by size (also topology) --Often used (with DNA size standards) to determine the size of DNA fragments. 1. Linear DNA fragments migrate through an agarose gel matrix with a mobility that is inversely
proportional to the log10 of their molecular weight. 2. DNA topology also affects migration through agarose gels.
--Closed circular DNA generally migrates more slowly than linear DNA. --Introduction of supercoils into circular DNA increases mobility --Fully supercoiled circular DNA actually migrates faster than linear DNA of the same size.
Agarose Gel Electrophoresis - Considerations
1. Agarose concentration
Gels of different concentrations are useful for resolution of DNA fragments of different sizes.
Agarose Gel Electrophoresis - Considerations
1. Agarose concentration
Gels of different concentrations are useful for resolution of DNA fragments of different sizes. 2. Voltage
Too high - can result in poor resolution of larger fragments, or even melt the gel. Too low - takes forever. Rule of thumb - 5 V/cm (distance between electrodes)
Agarose Gel Electrophoresis - Considerations
1. Agarose concentration
Gels of different concentrations are useful for resolution of DNA fragments of different sizes. 2. Voltage
Too high - can result in poor resolution of larger fragments, or even melt the gel. Too low - takes forever. Rule of thumb - 5 V/cm (distance between electrodes)
3. Buffer
Will not run in water - need a salt-containing buffer (ions for conductivity, buffer for maintaining pH and thus ions) Most common are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE)
Agarose Gel Electrophoresis - Considerations
1. Agarose concentration
Gels of different concentrations are useful for resolution of DNA fragments of different sizes. 2. Voltage
Too high - can result in poor resolution of larger fragments, or even melt the gel. Too low - takes forever. Rule of thumb - 5 V/cm (distance between electrodes)
3. Buffer
Will not run in water - need a salt-containing buffer (ions for conductivity, buffer for maintaining pH and thus ions) Most common are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE)
4. Visualization
Cannot see DNA by itself - must use dye to visualize it. Most common is ethidium bromide (EtBr) - add to gel at 0.5 ug/ml (can also soak gel in EtBr-containing solution after gel run). DNA intercalator; fluoresces when exposed to UV light, but 30X more fluorescent when intercalated in DNA.
Agarose Gel Electrophoresis - Considerations
1. Agarose concentration
Gels of different concentrations are useful for resolution of DNA fragments of different sizes. 2. Voltage
Too high - can result in poor resolution of larger fragments, or even melt the gel. Too low - takes forever. Rule of thumb - 5 V/cm (distance between electrodes)
3. Buffer
Will not run in water - need a salt-containing buffer (ions for conductivity, buffer for maintaining pH and thus ions) Most common are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE)
4. Visualization
Cannot see DNA by itself - must use dye to visualize it. Most common is ethidium bromide (EtBr) - add to gel at 0.5 ug/ml (can also soak gel in EtBr-containing solution after gel run). DNA intercalator; fluoresces when exposed to UV light, but 30X more fluorescent when intercalated in DNA.
5. Quantitation
Use comparison to defined DNA size standards to estimate size of DNA fragments in samples. Can also use defined DNA amounts to estimate amount of DNA present on gel by EtBr staining.
Agarose Gel Electrophoresis - Limits
--At more than 2-3% agarose, gels become opaque, resolving power not high enough regardless
to resolve fragments <100 bp very well. Solution: Use polyacrylamide. --At less than 0.5%, agarose gels are extremely sloppy and impossible to work with, not able to resolve
DNA fragments >15 kb very well. Solution: pulsed-field gel electrophoresis (PFGE).
Angle of anode and cathode changed periodically during the run. “Reptation” - movement of DNA through agarose gel matrix. Large fragments “caught up” or “stuck” in the matrix. Changing angle of current flow frees large fragments. Can resolve DNA fragments up to chromosome size.
Mg2+
The Importance of Mg2+ Optimization
Mg2+
The Importance of Mg2+ Optimization