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

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.


--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.


--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.



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)





3. Buffer


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.





3. Buffer


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

mdb lab 2 lecture agarose gels

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