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” (http://www.nature.com/scitable/definition/restriction-mapping-283 ) 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) 1. Student title page (on a separate page) 2. Tables (with number and titles) – each table on a separate page 3. Figures (with number and titles and a legend, if appropriate) – each figure on a separate page 4. Commentary – as indicated below. 5. Answers to questions (see below) – no page limit, but be brief! 6. References Instructions:
You will need to familiarize yourself with the use of the NEB cutter tool (http://www.labtools.us/nebcutter-v2-0/ ) Click the # viral and phage box and select lambda from the drop down menu for standard sequences. Select linear and NEB enzymes and click submit. 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 to show the number of fragments produced and the length of each fragment. Assignment 1 – Restriction Endonuclease Analysis of Lambda DNA using NEB cutter 2 Click view gel under main options. This show 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. 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 are as follows: For the in silico digestion using NEB cutter (http://www.labtools.us/nebcutter-v2-0/)
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) Tables showing the number of restriction sites for each of the digests listed above and the number of fragments generated in each digest A restriction map 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): A single map consist of a “line” for the double digest flanked by ‘lines” for each of the single digests (as shown in examples provided in tutorials), each line showing digestion sites and fragment sizes. 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!) A commentary (similar to the narrative provided in the example on mapping discussed in class) explaining how you went about constructing the map from the NEB cutter data.