Question
If you start with 10 µg of supercoiled plasmid and assume that your recovery efficiency is 100% in all steps, what is the final concentration of the 1.6 kb DNA you purify, if you elute with 100 µl of TE buffer?
Distances between restriction sites:
1 & 2: 1 Kb;
1 & 4: 1.1 kb;
2 & 3: 1.5 kb;
3 & 4: 4 kb
The following is a simple map of a plasmid. The distances between the restriction enzymes are indicated. Please design an experiment and describe conceptually how you would purify the 1 kb segment between the NcoI and EcoR I sites, from purified supercoiled plasmid DNA. If you start with 10 g of supercoiled plasmid and assume that your recovery efficiency is 100% in all steps, what is the final concentration of the 1.6 kb DNA you purify, if you elute with 100 l of TE buffer? Distances between restriction sites: 1 & 2: 1 Kb; 1 & 4: 1.1 kb; 2 & 3: 1.5 kb; 3 & 4: 4 kb
Explanation / Answer
A super coiled plasmid is given and the restriction profileof it is given as below- Distance between restriction sites is - NcoI and EcoRI = 1 kb NcoI and EcoRI = 1.1 kb EcoRI and BamHI = 1.5 kb BamHI and EcoRI = 4kb To obtain 1 kb segment between the NcoI and EcoR I sites of plasmid, the following restriction analysis is done - First cleave the plasmid with BamHI and NcoI which results in two fragments of each 5.1 kb and 2.5 kb. Electrophoresis of this fragments will result in separation of both fragments, and smaller fragment of 2.5 kb is taken. Now, digest it with EcoRI that results in the 1.5 kb and 1 kb fragments. Electrophoresis of these two fragments will resukt in their separation and smaller fragment is purified. NcoI--------------5.1 kb-----------BamHI BamHI ---------2.5 kb-------------NcoI Taken, BamHI ---------2.5 kb-------------NcoI BamHI ---------2.5 kb-------------NcoI BamHI -------1.5 kb -------EcoRI--------1 kb ------NcoI 10 µg of supercoiled plasmid is taken and 100% recovery efficiency is there. The size of this plasmid is 7.6 kb. The final concentration of the 1.6 kb DNA is 7.6 kb -------------> 10 µg 1.6 kb -----------> ? = 1.6 x 10 / 7.6 = 2.1 µg 10 µg of supercoiled plasmid is taken and 100% recovery efficiency is there. The size of this plasmid is 7.6 kb. The final concentration of the 1.6 kb DNA is 7.6 kb -------------> 10 µg 1.6 kb -----------> ? = 1.6 x 10 / 7.6 = 2.1 µg