Suppose you find a DNA plasmid in your lab freezer from years ago, but the label

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 out 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

Plasmid DNA of unknow sequence can be done by the two process using the restriction mapping and other is by the restriction digestion , gel electrophoresis insertring the unknown sequence into the vector for the expression of the gene .

First procedure ;

The plasmid of the unknown sequence is restriction digested with the two restriction enzymes with two unknown enzymes ,which will digest , these are then gel electrophorised and we get bands with two different bands ,If enzyme does not cut it will give bands of the different sizes . Then this unknown sequence is taken form the gel eletrophoresis .it is then incorperated into the shuttle vector with the known sequence in a multiple cloning site .these are transformed into the bacteria to grow large quantities of the vector with our unknown band .then it is sequenced through the polymerase chain reaction to amplify the DNA and by sequencing this DNA we will get to know the sequence .

or the unknow sequence can be restriction digested using the enzymes and gel elctrophoresris is done and then restriction mapping is done where we get the structural information of the the DNA .The distance between the restriction site is determined

Related Questions

Navigate

• Browse (All)
• Browse S
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.