Assignment Task
In this exercise you will perform a virtual digestion of lambda DNA using NEB cutter, an online tool containing a database of common DNA sequences.
Objectives
- To introduce students to a bioinformatics tool
- To demonstrate the effects of agarose percentage on DNA separation
- To introduce students to restriction mapping – “a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints”
Question
A) Gel electrophoresis (general questions)
1. If a linear piece of DNA has 2 recognition sites, how many bands would you expect to see on a gel after digestion with a restriction enzyme that recognizes each site?
2. If a circular piece of DNA has 2 recognition sites, how many bands would you expect to see on a gel after digestion with a restriction enzyme that recognizes each site?
3. If a liner DNA molecule is digested exactly in the middle, how many fragments would you expect to see on a gel? Explain your answer.
4. When electrophoresed on an agarose gel, will the largest DNA fragment be closest to or farthest from the loading well? Explain your answer.
5. If you wanted to resolve small DNA molecules (e.g. 100 – 1000 bp in length), would you use a higher agarose percent gel or a lower agarose percent gel? Why?
6. If you wanted to resolve larger DNA molecules (e.g. above 5000 bp in length), would you use a higher agarose percent gel or a lower agarose percent gel? Why?
B) In silico digestion using NEB cutter
7. How does the number of restriction sites for each digest relate to the number of fragments produced in the digest?
8. Add up the lengths of the fragments in each digest and verify that the total length matches that of the uncut lambda DNA.
9. Using NEB cutter, generate an EcoRI digest of linear lambda DNA. View the gel using 0.7% agarose, 1.4% agarose and 2% agarose. You can change the agarose % by changing the gel type in the “gel” window. How and why does the number and position of bands change as you increase the agarose percentage?
C) For Figure 1 below
10. Consider Figure 1 below. Does the experimental gel look like the virtual gel(s) generated by NEB cutter? If not, why not?
11. Count the number of bands in each lane of the experimental gel in Figure 1. How does the number of bands you counted compare to the number of bands generated in the corresponding NEB cutter digests? What could be a possible explanation for the deviation seen between the experimental digest and the in silico digest?
