Gel Electrophoresis
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| Electrophoresis allows for what on the basis of what? | It allows separation of molecules in an electrical field on the basis of size/molecular weight and shape.
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| A molecule with a negative charge is called a | anion
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| an anion will migrate toward what? also what is it called | the positive electrode, called an anode
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| a molecule with a positive charge is called a | cation
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| a cation will migrate toward what? and what is this called? | the negative electrode, called a cathode
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| the migration and separation of molecules are carried out using a what? also what are some examples of these? | carried out using a solid matrix, such as agarose and polyacrylamide
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| the solid matrix retards the movement of molecules by what is called a | seiving effect, in which small molecules navigate the matrix more quickly than larger ones
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| electrophoresis is used chiefly for analysis and purification of large molecules such as | proteins and nucleic acids
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| for now, we will be separating DNA fragments using ______ as the matrix | agarose
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| the relative mobility of the fragments, (how fast they travel through the matrix relatvive to each other), or the migration will depend on several parameters: | size, shape, % agarose, voltage
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| Regarding size: | smaller fragments of DNA will travel further than the larger ones
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| regarding shape: bacteria possess extrachromosomal circular DNA known as _____. ______ _______ come in three distinct shapes | plasmids; isolated plasmids
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| Regarding % agarose: fragments _______ than the matrix pore size cannot enter the gel and are not ________. At the other extreme, fragments ______ than the pore size are not ________ at all. | larger; not resolved; smaller; not retarded
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| what are two significant equations regarding voltage? | Ohms law (V=IR) and the power equation, which is P=VI
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| the _______the voltage, the _______ the power, which creates lots of ____ | the higher the voltage, the greater the power, which creates lots of HEAT
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| non-uniform heat distribution results in ___________ | smiling bands (heat is more rapidly dispersed at EDGES OF GEL)
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| what is the measurement of dispersion? | 5-8 cm/V
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| During electrophoresis water is_________, generating ________at the anode and ______ ______ at the cathode. This results in regions of high and low pH. Therefore a _______ is needed. | electrolyzed; protons; hydroxyl ions; buffer
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| the two most popular buffers for agarose gel electrophoresis are _____ and _______. | Tris-Borate-EDTA (TBE) and Tris-Acetate-EDTA (TAE)
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| what are two examples of loading dye? | glycerol and the dye bromophenol blue~200-400 bp)
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| Ethidium bromide is a dye that ________between the base of ______ _____ (DNA and EtBr travel in opposite orientation). When it does, there is a shift in its _________ ________ and it will _______ under UV light | intercalates; nucleic acids; absorption spectrum; fluoresce
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| _______ _________ is semi-quantitative | band intensity
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| a single band represents _______ _____________ _______ ______ | high molecular weight DNA
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| sheared DNA indicates poor quality DNA and is represented by _______ | a smear
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| the molecules separate by ______ RELATIVE TO THE CONDITIONS in which the electrophoresis was conducted | size
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| A ____ _________ (a sample of DNA fragments of known sizes and mass) is used as a ________ to estimate the size of unknown DNA molecules | DNA marker; reference
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