Experiment 5. Restriction Enzyme Digest and Plasmid Mapping. VY NGUYEN 26 February 2016

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1 Experiment 5 Restriction Enzyme Digest and Plasmid Mapping VY NGUYEN 26 February 2016 ABSTRACT 1. Understand the use of restriction enzymes as biotechnology tools 2. Become familiar with the principles and techniques of agarose gel electrophoresis 3. Estimate DNA fragment sizes from agarose gel data 4. Use a restriction map to predict how many fragments will be produced in a given restriction digest

2 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 1 1. The restriction digest map of puc19 is shown in Figure 4. Suppose you performed restriction digestions of puc19 by the various enzymes listed in Table 1 (each row represents a separate digestion) 1) predict the sizes of fragments in base pairs and write your answers in Table 1 EcoRI: one cut, so the whole plasmid remains the whole length Bsey I: one cut, so the whole plasmid remains the whole length Ava II: two cuts, so there will be 2 fragments = = 2464 EcoRI + Bsey I: two cuts,so there will be 2 fragments = = 1972

Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 2 EcoRI + Ava II: three cuts, so there will be 3 fragments 2059 1837 = 222 1837 396 = 1441 2686 1441 222 = 1023 BseyI + Ava II:

3 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 2 EcoRI + Ava II: three cuts, so there will be 3 fragments = = = 1023 BseyI + Ava II: three cuts, so there will be 3 fragments = = = ) Label the positions of the wells, label the lanes with the enzymes used. Include a lane with 500 bp DNA ladder. Draw the corresponding DNA fragments on the gel diagram, and label the size of each DNA fragment. Label the cathode and anode.

Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 3 2. Print the photograph of your gel from elearning. Label the wells, the lanes with the enzymes used.

4 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 3 2. Print the photograph of your gel from elearning. Label the wells, the lanes with the enzymes used. A 500 bp ladder containing 16 bands in 500bp increments (the smallest one is 500 bp) was used to estimate the sizes of DNA fragments on your gel. Note the sizes of the four smallest DNA fragments in the molecular ruler/ladder. Estimate and label the size of each fragment in each restriction digest sample. Calculate the sizes of fragments in base pairs based on the information provided in Fig 3 and label the size for each band (one set). Label the cathode and the anode. Estimated DNA fragment sizes

5 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 4 Calculated DNA fragment sizes Enzymes Fragments produced (bp) Calculation EcoRV 5371 PstI = = 4296 EcoRV+PstI = = = 2576

6 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 5

7 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 6 3. Visit the New England Biolabs website ( and click on NEB CUTTER at the bottom of the screen. Open the file of pglo sequence found at elearning. Copy and paste the sequence into NEBcutter. Make sure the sequence doesn t have linebreaks in it. Type pgloyour name BIOL2281 in Name of sequence. Select the sequence is circular. Select NEB enzymes and click submit. You will get the restriction map for pglo.

8 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 7 Then click on Custom Digest under Main Options. Choose EcoRV, PstI. Select Digest. It will display the restriction map with just these enzymes.

9 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 8 Click view gel under Main options. Print the page with a virtual gel and the list of fragments. Your name should be displayed on the title of the print out. On the page of Custom Digest, five of the open reading frames are identified on the plasmid map. Click on the curved bars of c. Click Blast this sequence at NCBI under the protein sequence and find out the name of the protein superfamily. Repeat the search steps for curved bar of b and find out the name of the protein superfamily. Protein superfamily for curve bar of c: GFP Protein superfamily for curve bar of b: transpeptidase

10 Experiment 5: Restriction Enzymes Digest and Plasmid Mapping Vy Nguyen 9 Experiment 5. Restriction Digestion and Mapping 1. Be able to identify DNA bands on a gel using DNA markers as references. okay 2. Define restriction endonucleases; understand the difference between Exonucleases and Endonucleases Restriction endonucleases : proteins that cut DNA at specific sites by recognizing specific sequences of DNA base pairs and cut, or chemically separate, DNA at that specific arrangement of base pairs. Endonucleases: Endonuclease enzymes are enzymes that cleave to the bonds of the DNA from within the molecule. Exonucleases : Exonuclease enzymes are a category of enzymes that cleave to the nucleotides at the ends of the DNA molecule. 3. What is a palindromic sequence? A palindromic sequence is the sequence of bases that reads the same forwards as it does backwards on the opposite DNA strand 4. Be able to list the essential components of a restriction digest reaction. DNA An appropriate buffer The restriction enzyme Deionized water 5. Given a plasmid map with known restriction sites and the enzyme or combinations of enzymes used in a restriction digest, be able to determine the sizes of restriction fragments produced in the digest. Approximately draw the DNA bands on a gel diagram according to the marker lane. ok 6. Be able to choose an appropriate concentration of agarose gel to obtain a good separation of DNA. 500bp 10,000bp > 0.8 1% of agarose >10,000 bp > 0.5% of agarose Higher percentage gel is better with lower bp 7. Know how to prepare a given percentage of agarose in a given volume of buffer. (e.g. how to make a 1.5% agarose gel dissolved in a final volume of 50 ml of buffer) 1.5% agarose gel = 1.5 g of agarose/100 ml of buffer=(? g of agarose /50 ml of buffer).75 g of agarose in 50 ml of buffer 8. When will you add the loading blue dyes to the restriction enzyme digested sample? How is DNA visualized? After extracting the centrifuged samples from the 37 celcius water bath DNA is visualized on a UV light box

Chapter 11. Restriction mapping. Objectives

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