Question

In your previous prac session you digested your POTC-A plasmid DNA with 3 enzyme mixes (AB and C). One mix contained the restriction endonuclease Kpnl alone, another contained both del and Not and another contained Sphi and Nhe, but you don't know which was which yet. The sites where these enzymes cut POTC-A are indicated on the plasmid map at the top of the page. Calculate the sizes of the DNA fragments that you would expect to see on your agarose gel following a restriction digest with each of these three restriction enzyme combinations, Marking of this question will be automated, so it is essential that you adhere to the following rules regarding the format of your answers, otherwise they will be marked as incorrect:

Answer 1

i) KpnI alone:

From the plasmid map, we find that there are two restriction cut sites of KpnI on the plasmid. One at 988 bp position & other at 1601 bp position. When the plasmid is mixed with KpnI alone, KpnI will cut the plasmid at 988 & 1601 bp positions. As a result, we will find two fragments. The fragment lengths will be 613 bp (Calculated by subtracting 988 from 1601) & 5788 bp (Calculated by subtracting 613 from total plasmid size 6401).

ii) NdeI & NotI:

From the plasmid map, we find that there are two restriction cut sites of NdeI on the plasmid. One at 484 bp position & other at 3369 bp position. There is one restriction cut site of NotI at 2310 bp position. When the plasmid is mixed with NdeI & NotI, NdeI will cut the plasmid at 484 & 3369 bp positions; NotI will cut the plasmid at 2310 bp position. As a result, we will get three fragments- one from NdeI (484 bp) to NotI, another from NotI to NdeI (3369 bp) & another between two NdeI cut sites. Thus, fragment lengths will be 1826 bp (Calculated by subtracting 484 from 2310), 1059 bp (Calculated by subtracting 2310 from 3369) & 3516 bp (Calculated by subtracting 1826 & 1059 from total plasmid size 6401).

iii) SphI & NheI:

From the plasmid map, we find that there are two restriction cut sites of NheI & SphI at 1569 bp & 2559 bp respectively. When the plasmid is mixed with SphI & NheI, NheI will cut the plasmid at 1569 bp position; SphI will cut the plasmid at 2559 bp position. As a result, we will find two fragments. The fragment lengths will be 990 bp (Calculated by subtracting 1569 from 2559) & 5411 bp (Calculated by subtracting 990 from total plasmid size 6401).