Modern Experimental Biochemistry

Chemistry

Quiz 15 :
The Action of Restriction of Endonucleases on Plasmid or Viral DNA

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Quiz 15 :
The Action of Restriction of Endonucleases on Plasmid or Viral DNA

In this problem, we are asked the way in which the EDTA buffer stops the restriction enzyme reaction. EDTA, an abbreviation for ethylene diamine tetra acetic acid, binds metal ions very effectively; it works as a chelating compound. Restriction enzymes need magnesium ions to function properly. EDTA chelates these metal ions out of solution, making them unavailable to support the activity of the enzyme, thus stopping the process.

In this problem, we are asked why we would use glycerol and bromo phenol blue in our buffer for our polyacrylamide gel. Glycerol is a viscous sugar-alcohol; it makes the sample thicker and stickier, thus easing loading onto the gel lanes. Bromo phenol blue is a tracking dye; most substances loaded onto a gel cannot be seen with the naked eye as they move through the gel. The bromo phenol blue can be seen during operations and thus we can see how the gel is progressing.

In this problem, we are asked to describe the polyacrylamide gel (PAGE) that would result from the digestion of ? phasge using the restriction endonuclease Eco RI. The phage in question is linear and double-stranded and has more than 48,000 base-pairs. Eco RI is a nuclease derived from E. coli , and it cleaves this phage into six fragments. Therefore, a PAGE run separating these fragments would have six bands.