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” Note: this assignment is not a formal manuscript. Please follow the instructions below.

Your assignment must

• Be double spaced
• Use 12 pt. Times New Roman font
• Have 1” margins (top, bottom, left and right)
• Have numbered pages
• Be your own work and in your own words

Your assignment should include the following items (in the order listed)

• Student title page (on a separate page)
  1. Tables (with number and titles) – each table on a separate page
  1. Figures (with number and titles and a legend, if appropriate) – each figure on a separate page
  1. Commentary – as indicated below.
  1. Answers to questions (see below) – no page limit, but be brief!
  • References

Instructions:

You will need to familiarize yourself with the use of the NEB cutter tool

• Click the # viral and phage box and select lambda NEB from the drop down menu
• Click on additional prefernces and select NEB enzymes and type I and II nucleases (not type III, and homing nucleases, or nicking enzymes)
• click save preferences, submit and then continue
• Select custom digest.
• Select the restriction enzymes needed by clicking the checkbox to the left of the enzyme’s name. The table shows the number of sites, the recognition sequences, and % activity of the enzyme in different NEB reaction buffers.
• Click digest at the bottom of the screen. The results show the linear lambda DNA with the enzyme’s restriction sites (scroll over them to reveal the position of the restriction site).
• Click fragments under the heading list at the top to show the number of fragments produced and the length of each fragment.

• Click gel under main options at the top. This shows you the fragments on a virtual gel. You can vary the % of agarose to see how it would affect the appearance of the bands on the gel (this can be done by clicking gel % under specifications above the virtual gel).
• Perform in silico digestions using (1) EcoRI (single digest), (2) BamHI (single digest),

(3) HinDIII (single digest), (4) EcoRI + BamHI (double digest) and (5) EcoRI + HinDIII (double digest) of linear lambda DNA.

The results/analyses that should be included in your assignment are as follows:

1. For the in silico digestion using NEB cutter
   • 1-1 Tables showing fragment sizes generated by single (EcoRI, BamHI, HinDIII) and double (EcoRI + BamHI and EcoRI + HinDIII) digests (you can show a separate Table for each digest or combine results into one Table – that’s up to you)

• 1-2 Tables showing the number of restriction sites for each of the digests listed above and the number of fragments generated in each digest

·       1-3 Restriction maps for the EcoRI sites relative to the BamHI using the in silico digestion (NEB cutter) data (make it a figure – include an appropriate figure title below the map, and a legend, as needed):

• For 1-3: Include the following: A map (“line”) for the double digest, with maps (“lines”) for each of the single digests (as shown in examples provided in tutorials) above and below the double digest map (shown in the lamda digestion example on D2L). Show the numerical position of digestion sites and the fragment sizes between the digestion sites. It is expected that you construct the map based on the in silico digestion results (i.e. do not just cut and paste the map produced by NEB cutter!)

• Therefore, you need to produce three (3) maps (lines):

• 1) Lambda DNA with EcoRI digestion sites and fragment lengths between the digestion sites

• 2) Lambda DNA with EcoRI/BamII digestion sites and fragment lengths between the digestion sites

• 3) Lambda DNA with BamII digestion sites and fragment lengths between the digestion sites

• Note that the digestions sites in the maps (lines) listed above should line up: the EcoRI digestion sites in the single digest should line up with the EcoRI digestion sites in the double digest and the BamII digestion sites in the single digest should line up with the BamII digestion sites in the double digest.

Assignment, continued

• For the gel provided in Figure 1 below include a:

• 2-2 Table showing the number of fragments shown in the lanes for each of the single (EcoRI, BamHI, HinDIII) and double (EcoRI + BamHI and EcoRI + HinDIII) digests. It’s best if you count the fragments while looking at the figure on a computer screen – some bands are faint and may be difficult to see if you print the figure.

Questions/tasks:

• 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?
  1. 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?
  1. 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.
  1. When electrophoresed on an agarose gel, will the largest DNA fragment be closest to or farthest from the loading well? Explain your answer.
  1. 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?
  • 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?

• In silico digestion using NEB cutter:
  • How does the number of restriction sites for each digest relate to the number of fragments produced in the digest?
  • Add up the lengths of the fragments in each digest and verify that the total length matches that of the uncut lambda DNA.
  • 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?

• For Figure 1 below:
  • Consider Figure 1 below. Does the experimental gel look like the virtual gel(s) generated by NEB cutter? If not, why not?
  • 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?

Fig. 1: Restriction endonuclease digestion of linear lambda DNA. Fragments originating from single and double digests were electrophoresed on 0.7% agarose gel. Lambda DNA digested with BamHI (lane 1), BamHI/ EcoRI (lane 2), EcoRI (lane 3), EcoRI/ HinDIII (lane 4), HinDIII (lane 5) and HinDIII/ BamHI (lane 6). Markers (lambda DNA digested with EcoRI and EcoRI/ HinDIII) are shown in lanes 7 and 8, respectively).

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