Question

III. Subclone the gene into plasmid, extract the plasmid DNA. 5. You know that your insert (gene of interest, GOI) is flanked by the EcoRI sites, which makes this restriction enzyme a perfect candidate to cut out your gene. You also know that the GOI has a unique BamH1 restriction site. After subcloning the PCR product into the plasmid, a purified DNA preparation of the plasmid is digested to completion with BamHI restriction endonuclease. In separate reactions, the same preparation was digested to completion with EcoRI and with a mixture of EcoRI and BamHl, respectively. The diagram below shows the appearance of the original molecules and the digestion products in the three digestion mixtures, after separation by electrophoresis in an agarose gel. Lane M-Linear DNA markers of the indicated length in kb M #1 #2 #3 #4 Lane 1 - Sample of original plasmid DNA preparation Lane 2 - Sample of the products of BamHi digestion Lane 3 - Sample of the products of EcoRI digestion Lane 4 - Sample of the products of digestion of BamHI + EcoRI ILITI TIT a) What was the original DNA molecule's length? b) Was the original plasmid DNA circular or lincar? c) How many cuts does the Eco RI make?

Answer 1

Ans- on looking the bands on agarose gel we find that in lane 1 in which sample of original plasmid DNA is placed the band is between 2kb and 3 kb of marker band so the original DNA length was approx. 2.5 kb.

The lane in which sample with BamHI digestion is placed only one band of 9kb present. So if the plasmid DNA is linear and is digested at one place there will be 2 bands but if the plasmid is circular then digestion at one place will produce only one band.

• When the plasmid DNA is linear and digested at one place Two fragments of DNA produced. • When the plasmid DNA is circular and cleaved at one place Only one fragment produced

In lane 3 in which sample digested with EcoRI is placed have two bands, so as we seen earlier that if the plasmid is circular then single cut produces single band, so for two bands digestion at two places required.

When the circular plasmid DNA is digested at two places Two fragments are produced

Hence our answers are-

A- original DNA length is approx. 2.5 kb

B- original plasmid DNA is circular because on 1 cut it produces on band on agarose gel

C- EcoRI made 2 cuts. Because two bands are present on the agarose gel and when circular DNA is cleaved at two places then only two bands will be produced.