Techniques of Molecular Biology Flashcards
Gel electrophoresis separates proteins based on what?
Size and shape.
What are the two types of gel matrices that are used in electrophoresis procedures?
Polyacrylamide and agarose.
What gel matrix has high resolving power but separates in very low size ranges?
Polyacrylamide
What gel matrix has low resolving power but separates in very high size ranges?
Agarose
Why is it that DNA above 40 kb can’t be resolved using normal gel electrophoresis?
Because after a certain point, the fragments can’t fit through the pores of the matrix and migrate at the same rate.
Explain how pulse-field gel electrophoresis works?
Uses a pair of cathodes located at the top corners and a pair of anodes located at the bottom corners of the gel and applies “pulses” of alternating electric fields at right angles to each other. The DNA sample will move towards the anode, but the fragments will be resolved by size because larger fragments will take longer to orientate themselves in the direction of the electric field in between pulses.
What is the advantage of pulse-field gel electrophoresis compared to normal gel electrophoresis?
It’s able to resolve DNA molecules that are several megabases in length, whereas normal gel electrophoresis can only resolve molecules that are below 40 kb and even less if polyacrylamide is being used.
A sample of bacterial DNA contains the same molecule of DNA in linear, circular, and supercoiled forms. What would the gel look like after an electrophoresis procedure was performed on it? Justify your answer.
There would be three bands. The topmost band would be the circular molecule, followed by the linear and then supercoiled molecule. Shapes that take up less volume migrate through the gel faster than those that take up more volume.
What property of RNA prevents it from being used in electrophoresis without the use of special reagents? What do these reagents do to the molecule?
RNA is capable of forming complex secondary and tertiary structures which adds another variable that could influence their mobility in the gel. The reagents will prevent RNA from forming hydrogen bonds so that the mobility of the molecule is proportional to it’s molecular weight.
What is DNA cloning?
When you insert recombinant DNA into a host cell so that the host can propagate it.
What is recombinant DNA?
DNA made from
What is DNA cloning?
When you insert recombinant DNA into a host cell so that the host can propagate it.
What is recombinant DNA?
DNA made from
