Molecular Scissors: Lambda Digest Teacher Materials

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1 Molecular Scissors: Lambda Digest Teacher Materials Students will conduct a restriction digest of lambda DNA using two unknown enzymes. They will use the results of gel electrophoresis to identify the enzymes as HindIII or EcoRI based on the band patterns generated by the digests. Learning Goals, Objectives, and Skills... 2 Instructor Planning Guide... 3 Instructor Preparation Guide... 5 Answers to Student Questions... 7 Standard Alignments.10 Calculation Tool for ordering NEB Reagents. 14 Last updated: August 7, 2017

2 Student Learning Goals: Molecular Scissors Learning Goals Students will understand what a restriction enzyme is and what it does. Students will understand the process of agarose gel electrophoresis. Students will identify a need for DNA restriction analysis. Students will understand how to use restriction enzyme analysis to determine a general restriction map. Student Learning Objectives: Students will plan a restriction enzymes analysis that will provide meaningful data. Students will use restriction enzymes as biotechnology tools. Students will perform the technique of agarose gel electrophoresis. Students will estimate DNA fragments sizes from agarose gel data. Students will analyze the results of the molecule separation by gel electrophoresis. Students will identify two unknown restriction enzymes based upon their data. Scientific Inquiry Skills: Students will pose questions and form hypotheses. Students will design and conduct scientific investigations. Students will use experimental data to make conclusions about the initial question and to support or refute the stated hypothesis. Students will follow laboratory safety rules and regulations. Laboratory Technical Skills: Students will demonstrate proper use of micropipettes. Students will consider safety considerations when working with an electric current. Students will demonstrate proper use of gel electrophoresis equipment. Students will prepare and pour agarose gels. Students will perform restriction enzyme digests. 2

3 Experimental Timing Tips: Molecular Scissors Instructor Planning Guide From start to finish this lab takes minutes. However, there are many good stopping points in this protocol that make it possible to complete the lab in a series of minute periods. Pre-lab discussion (20-30 min) Restriction digest setup (15 min) and incubation (15-30 min) Stopping Point - Reaction tubes can be placed in the freezer after the restriction digest has been performed. Tubes can be frozen until you are ready to perform the gel electrophoresis. Frozen tubes can be stored almost indefinitely. Preparation of agarose gels and buffer (30 min)* Electrophoresis of DNA (25 min)** Visualization and interpretation of gels (10-15 min) *Instructors may choose to prepare gels and buffer ahead of time to reduce lab time. **Time required for electrophoresis may vary depending on the type of equipment and voltage used. Specialized Equipment: p20 micropipettes gel electrophoresis units with power supplies (each student group will run three samples plus a ladder) transilluminator or other UV light source water bath or incubator centrifuge (optional) Ordering information: This lab was developed using Fisher Scientific products and reagents (DNA ladder, lambda and restriction enzymes) from New England BioLabs. The reagents from New England BioLabs can be ordered (at no cost) by going to their website ( A calculation tool for ordering NEB Reagents for this lab can be found on the final page of this document. 3

4 Procedure Tips: 1. Before starting the experiment, ask students to check their materials list to make sure they have everything. 2. Demonstrate how to pipet very small volumes of liquid and how to load a gel. If your students have not had the opportunity to run many gels, you may want to have them practice loading gels before you begin this experiment. You can make practice gels by melting down old gels. Practice gels can be removed from the gel trays and stored in a plastic bag with a damp paper towel or small amount of water in the refrigerator for weeks. Students can practice using dilute loading dyes. 3. Remind students to use a fresh pipette tip between each addition when setting up the restriction digest and when loading each sample into the gel. 4. Remind students to record the location of each of the samples as they load them into the gel. 5. Remind students to keep all their reagent tubes on ice. Teaching Tips: 1. The protocol for preparing the electrophoresis gels is not included in this version of the lab. You can download the gel document from the website ( adapt it to your equipment and insert it in the lab. 2. Restriction maps of Lambda/HindIII and Lambda/EcoRI can be found on page 13 of this document. New England BioLabs has many useful interactive tools on the NEB website Safety Considerations: Gloves, lab coats and eye protection should be used whenever possible as part of good laboratory practice. Practice sterile techniques whenever possible, to avoid contamination of reagents. Exercise caution when heating and/or melting reagents during gel preparation. Exercise caution when working with electrical equipment. UV protective shields and/or glasses must be used if visualizing gels with a UV transilluminator. Always wash hands thoroughly after handling biological materials or reagents. Obtain the Material Safety Data Sheets (MSDS) available from the suppliers, and follow all safety precautions and disposal directions as described in the MSDS. Check with your school s lab safety coordinator about proper disposal of all reagents and gels containing DNA stains. 4

5 Molecular Scissors Instructor Preparation Guide Materials: This guide assumes 30 students, working in groups of two, for a total of 15 groups. Advanced Teacher Preparation: 1 tube of lambda DNA 500 g /L (NEB # N3011S ). 1 tube will have 250 g DNA in 500 L solution. 1 tube of EcoRI-HF (NEB # R3101S) 1 tube contains 500 L of enzyme 1 tube of HindIII-HF (NEB # R3104S) 1 tube contains 500 L of enzyme 1 tube 10X concentrated CutSmart buffer (provided with enzymes) 1 tube Quick Load 1 kb Extend Ladder (NEB # N3239S) 1 tube will contain 1.25 ml of DNA at 20 g/ml 1 ml gel loading dye (provided with enzymes) dh 2 O 100 microcentrifuge (1.5mL) tubes Electrophoresis materials and equipment 15 ice buckets or styrofoam cups with crushed ice 15 p20 micropipette and pipette tips 15 microcentrifuge tubes with 200 L sterile water 15 tubes with 20 L CutSmart buffer 15 tubes with 10 L lambda DNA (500 ng/l) 15 tubes with 2 L restriction enzyme A (labeled enz A)* 15 tubes with 2 L restriction enzyme B (labeled enz B)* 15 tubes control (empty) 15 tubes with 10 L Quick Load 1kb Extend Ladder 15 tubes with 30 L loading dye 15 agarose gels (0.6%) with DNA stain 15 fine point permanent marker (such as Sharpie) 37 C water bath or incubator 65 C water bath or incubator UV or blue light transilluminator (appropriate for the DNA stain) Student Workstation: 1 ice bucket or Styrofoam cup with crushed ice 1 p20 micropipette and pipette tips 1 microcentrifuge tube rack 1 tube with 200 L water 1 tube Control (empty) 1 tube CutSmart buffer with 20 L buffer 1 tube lambda DNA with 10 L DNA (500 ng/l) 1 tube Enzyme A with 2 L restriction enzyme 1 tube Enzyme B with 2 L restriction enzyme 1 tube DNA Ladder with 10 L Quick Load 1kb Extend Ladder 1 tube loading dye with 30 L 6X dye 1 agarose gel (0.6%) with DNA stain 1 electrophoresis unit with power supply 1 extra fine point permanent marker Common Workstation: 37C water bath or incubator 65C water bath or incubator microcentrifuge (optional) UV light or blue light transilluminator 1X electrophoresis buffer 5

6 Easy Substitutions: Styrofoam cups can be substituted for ice buckets at student workstations. If you do not have a centrifuge, have students gently tap the microcentrifuge tubes on the lab bench to collect all the reagents at the bottom of the tube. Set-up Calendar: 2 weeks before lab Check supplies and order any needed materials. If making any substitutions to the supply list, edit the student protocol accordingly. 1 day before lab Set up student lab stations with all durable materials. Prepare TAE or buffer of your choice Aliquot out the lambda DNA, ladder DNA, loading dye, dh 2 O, buffer. Keep all tubes on ice or in freezer. Caution: Enzymes are heat sensitive. Keep all reagent tubes on ice. Morning of lab Prepare 0.6% agarose gel mix with DNA Stain. Pour agarose gels (1 gel/group) Aliquot 2 L of EcoRI in tubes labeled Enz A and 2 L of HindIII into tubes labeled Enz B. Keep all tubes on ice. 6

7 Pre-Lab: Molecular Scissors Answers to Student Questions 1. Restriction enzymes (or restriction endonucleases) are enzymes found in bacteria that cut DNA at specific sequences called restriction sites. 2. The bacteria are able to produce restriction enzymes that recognize sites on the T-4 viral DNA and cut the T-4 DNA into small non-infectious fragments CGATCG 3 3 GCTAGC 5 4. EcoRI would be the better choice because it produces sticky ends. HaeIII produces blunt ends that are harder to join together. 5. Lane 1. A double digestion with HindIII and BamHI will produce three bands: one long and two shorter bands. Lane 4 illustrates a single digest with HindIII, and Lane 3 shows a single digest with BamHI. In-Lab: Table 3: Single digest restriction fragments of Lambda DNA cut with EcoRI and Hindlll. Restriction Fragments HindIII Fragments in descending order Restriction Fragments EcoRI Fragments in descending order 23,130 23,130 21,226 21,

8 Data Collection Worksheet: Sample Gel: Lane 1: Blank Lane 2: Control Lane 3: Enz A (EcoRI) Lane 4: Enz B (HindIII) Lane 5: Ladder Lane 6: Blank Determine which enzymes were actually EcoRI and HindIII. Record it below. Enzyme A EcoRI Enzyme B HindIII The restriction site for EcoRI is GAATTC. Based on your results, how many times does the sequence occur in the lambda sequence? 5 Are the results consistent with what you expected? Are any bands missing? Can you suggest reasons why there is this discrepancy? Most students will be able to see 5 bands on the EcoRI digest. Because the 5804bp and 5643bp bands are so close in size, they will run together and appear as a single brighter, thicker band. If we were to use a higher concentration gel and/or run the gel longer, we would begin to see more separation between these two bands. Note: Students are unlikely to see the 564bp band on the HindIII digest. What is the total length of the Lambda DNA? 48,502bp 8

9 Post-Lab: DNA Standard Size (bp) of DNA Distance (mm) moved from well 23,000 Skip this band very large bands will skew the standard curve Unknowns Band Size (bp) of Distance (mm) moved DNA from well A , B , C , , ,

10 Molecular Scissors Standards Alignments MA Science and Technology/Engineering Standards High School (2016) Biology HS-LS1-1. Construct a model of transcription and translation to explain the roles of DNA and RNA that code for proteins that regulate and carry out essential functions of life. Chemistry HS-PS1-3. Cite evidence to relate physical properties of substances at the bulk scale to spatial arrangements, movement, and strength of electrostatic forces among ions, small molecules, or regions of large molecules in the substances. Make arguments to account for how compositional and structural differences in molecules result in different types of intermolecular or intramolecular interactions. HS-PS1-11(MA). Design strategies to identify and separate the components of a mixture based on relevant chemical and physical properties. HS-PS2-6. Communicate scientific and technical information about the molecular-level structures of polymers, ionic compounds, acids and bases, and metals to justify why these are useful in the functioning of designed materials. Physics HS-PS3-5. Develop and use a model of magnetic or electric fields to illustrate the forces and changes in energy between two magnetically or electrically charged objects changing relative position in a magnetic or electric field, respectively. NRC Practices Asking questions and defining problems Planning and carrying out investigations Analyzing data Mathematical and computational thinking Constructing explanations and designing solutions Engaging in argument from evidence Obtaining, evaluating, and communicating information Next Generation Science Standards High School (2013) Life Sciences HS-LS1-1. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins that carry out the essential functions of life through systems of specialized cells. Chemistry HS-PS1-3. Cite evidence to relate physical properties of substances at the bulk scale to spatial arrangements, movement, and strength of electrostatic forces among ions, small molecules, or regions 10

11 of large molecules in the substances. Make arguments to account for how compositional and structural differences in molecules result in different types of intermolecular or intramolecular interactions. HS-PS2-6. Communicate scientific and technical information about the molecular-level structures of polymers, ionic compounds, acids and bases, and metals to justify why these are useful in the functioning of designed materials. Physics HS-PS3-5. Develop and use a model of magnetic or electric fields to illustrate the forces and changes in energy between two magnetically or electrically charged objects changing relative position in a magnetic or electric field, respectively. Common Core State Standards Connections: ELA/Literacy - RST RST RST RST RST RST WHST WHST WHST WHST WHST SL Mathematics - MP.2 Translate quantitative or technical information expressed in words in a text into visual form (e.g., a table or chart) and translate information expressed visually or mathematically (e.g., in an equation) into words. Assess the extent to which the reasoning and evidence in a text support the author s claim or a recommendation for solving a scientific or technical problem. Cite specific textual evidence to support analysis of science and technical texts, attending to important distinctions the author makes and to any gaps or inconsistencies in the account. Integrate and evaluate multiple sources of information presented in diverse formats and media (e.g., quantitative data, video, multimedia) in order to address a question or solve a problem. Evaluate the hypotheses, data, analysis, and conclusions in a science or technical text, verifying the data when possible and corroborating or challenging conclusions with other sources of information. Synthesize information from a range of sources (e.g., texts, experiments, simulations) into a coherent understanding of a process, phenomenon, or concept, resolving conflicting information when possible. Write arguments focused on discipline-specific content. Write informative/explanatory texts, including the narration of historical events, scientific procedures/ experiments, or technical processes. Develop and strengthen writing as needed by planning, revising, editing, rewriting, or trying a new approach, focusing on addressing what is most significant for a specific purpose and audience. Conduct short as well as more sustained research projects to answer a question (including a self-generated question) or solve a problem; narrow or broaden the inquiry when appropriate; synthesize multiple sources on the subject, demonstrating understanding of the subject under investigation. Draw evidence from informational texts to support analysis, reflection, and research. Make strategic use of digital media (e.g., textual, graphical, audio, visual, and interactive elements) in presentations to enhance understanding of findings, reasoning, and evidence and to add interest. Reason abstractly and quantitatively. 11

12 MP.4 HSF-BF.A.1 HSF-IF.C.7 HSN.Q.A.1 HSN.Q.A.2 HSN.Q.A.3 HSS-IC.A.1 HSS-IC.B.6 Model with mathematics. Write a function that describes a relationship between two quantities. Graph functions expressed symbolically and show key features of the graph, by hand in simple cases and using technology for more complicated cases. Use units as a way to understand problems and to guide the solution of multi-step problems; choose and interpret units consistently in formulas; choose and interpret the scale and the origin in graphs and data displays. Define appropriate quantities for the purpose of descriptive modeling. Choose a level of accuracy appropriate to limitations on measurement when reporting quantities. Understand statistics as a process for making inferences about population parameters based on a random sample from that population. Evaluate reports based on data. 12

13 13

14 Calculation tool for ordering NEB Reagents for: Molecular Scissors Please keep in mind that NEB is a fantastic and generous partner and will provide up to $1000 of reagents for each school. Please check with your colleagues to coordinate your ordering to ensure that your school plans ahead for ALL of the planned labs requiring NEB reagents, and please, only order as much as you need. The calculation tool below will help you determine how much of each reagent to order. Importantly, the amount needed per group shown below includes the extra needed in case of mistakes or when aliquots are provided for each group. Fill out the chart below to determine how many tubes of each of the reagents you need to order. The following are important to keep in mind: The number of groups will vary depending on your classes and equipment. CutSmart Buffer and Loading Dye come with each restriction enzyme you order. Calculation tool: Example NEB Reagent NEB Catalog # Amount of Reagent In NEB Tube Amount Needed per Group Total Number of Groups Doing the Lab Total Amount You Will Need # Tubes Needed Reagent X X L 4 L 8 32 L 1 You fill this in 4 L X (# groups) 32 L < 40 L NEB Reagent NEB Catalog # Amount of Reagent In NEB Tube Amount Needed per Group Total Number of Groups Doing the Lab Total Amount You Will Need # Tubes Needed Lambda DNA N3011S 500 L 12 L EcoRI-HF R3101S 500 L 2 L HindIII-HF R3104S 500 L 2 L Quick-Load 1 kb Extend Ladder CutSmart Buffer Gel Loading Dye, Purple (6X) N3239S 1250 L 10 L* Provided with restriction enzymes Provided with restriction enzymes *This is per gel. You may have more than one group per gel. Once completed, you can submit your order here: 14

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