Tricky Question: Suppose you find a DNA plasmid in your lab freezer from years a

Question

Tricky Question: Suppose you find a DNA plasmid in your lab freezer from years ago, but the label has rubbed off the tube and you have no idea what plasmid it is. You perform spectrophotometry just to make sure it absorbs light at 260 nm wavelength (as DNA does), and you run it on an agarose gel to determine the approximate length of this circular DNA molecule (about 5000 bp). You wish to sequence this plasmid to identify it, but this poses a problem because you need some known sequence in a plasmid in order to design a sequencing oligonucleotide. Describe a strategy you will employ for sequencing this plasmid. Assume that you have access to standard molecular biology reagents (e.g.various restriction enzymes, bacteria, antibiotics, agarose and gel electrophoresis rigs) and that you have a contract with an outside company that will synthesize oligonucleotides of any sequence you request.

Explanation / Answer

Since restriciton enzymes can be used, a specific enzyme can be used. Then we will know that that specific restriction sequence is present in the plasmid. We can use this restriction site to create oligonucleotides for primers. These primers can then be used to decode the rest of the plasmid sequence.

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