Genetics Lab report

BIO 224 L. Hollis-Brown

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LAB 9: RESTRICTION ENZYME DIGEST AND GEL ELECTROPHORESIS OF DIGEST PRODUCTS

OBJECTIVES 1. Define restriction endonuclease. 2. Perform a restriction enzyme digest pf pMZ379. 3. Run a gel electrophoresis on the products of the restriction enzyme digest. 4. Determine the lengths of fragments created by the digest, using a standard curve. 5. Explain the purpose of the restriction enzyme digest of pMZ379 in the context of

CRISPR/Cas gene editing in fission yeast. BEFORE LAB 1. Read the lab handout in its entirety. If you do not prepare adequately for lab,

you will not be able to complete the lab in the time allotted. 2. Watch the video on the protocol for a restriction enzyme digest (also linked on D2L.) 3. Complete the pre-lab quiz before lab.

LAB SAFETY 1. Wear gloves and safety glasses throughout the procedure. 2. Clean your workstation before and after the procedure. 3. Wear closed-toed shoes and secure loose hair and clothing. 4. Dispose of reagents and used materials in labeled containers only. 5. Wash your hands after handling bacteria, even if you have been wearing gloves.

GENERAL NOTES ON LAB 1. Be sure to ask questions if you are unsure of any instructions.

2. Take careful notes as you do the lab. You will need these notes to write your final paper at the end of the semester.

3. You will turn in this lab handout for a grade. Be sure you have all questions answered in the handout. Also, include a picture of your gel, your standard curve, and all calculations.

Introduction This step uses a restriction enzyme digest to create distinguishable DNA fragments to confirm that the sgRNA has been inserted. Restriction enzymes recognize specific DNA sequences (the recognition site) and will cut the DNA at that location. The map of recognition sites is shown for the pMZ379 plasmid in Figure 1. This lab will use the restriction enzyme CspCI. This site is present in the original plasmid, but if the sgRNA is inserted during the PCR, the site is removed. Plasmids are circular and when inside

BIO 224 L. Hollis-Brown

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the cell are found in a supercoil structure. This structure, which is much like a rubber band twisted between one’s fingers until it becomes a ball, makes the plasmid small enough to fit inside of the E. coli cell. This also means that on an agarose gel electrophoresis, its balled-up structure makes it move faster than it would if it was a linear piece of DNA. Cutting the DNA with the CspCI enzyme will result in the creation of linear plasmid DNA. It is therefore expected that plasmid DNA cut with CspCI will run slower and appear larger than the uncut DNA, even though they are almost the same number of base pairs. This pattern seen on a gel electrophoresis will determine whether or not the plasmid has the sgRNA inserted, as shown in Figure 2 below.

Materials Per Group

Quantity Item plasmid preps isolated in Lab 8 4 1.5 mL microfuge tubes 1 plastic tube rack 1 ea. 2, 10, 20, 200 pipettes 1 box ea. sterile pipette tips, micro and small 300 μL sterile, nuclease-free water 30 μL aliquot CutSmart Buffer (5x) 5 μL aliquot SAM 5 μL aliquot CspCI 1 foam cooler for ice 1 bottle ethanol, for disinfecting 1 250 mL beaker for disposal of tips and tubes 1 permanent marker, fine

Shared by Class

Quantity Item 2 heat blocks or a water bath set at 37OC 1 ice bucket with ice 1 box ea. non-latex gloves, small, medium, large safety glasses 3 agarose gels with 10 wells and set-ups for running gels 2 bottles 1X TAE or equivalent running buffer

BIO 224 L. Hollis-Brown

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Figure 1. pMZ379 restriction map.

Protocol

Restriction Enzyme Digest

1. Keep all materials on ice.

2. Wear gloves and safety glasses.

3. Disinfect workstation and instruments with ethanol, DNase Away, if available.

4. Thaw the two tubes of recovered plasmids, and one regular plasmid, on ice.

5. Label five sterile microfuge tubes, one for each: no DNA, no enzyme, pMZ379, sgRNA tube 1 and sgRNA tube 2.

6. Place tubes in rack on ice. Arrange tubes in an orderly fashion and move tubes to a new rack as you use them.

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7. Add the components in the order shown in the table below. After adding each component, mark it off on the table.

Tube 1 Tube 2 Tube 3 Tube 4 and 5

Component No DNA No Enzyme

(pMZ379 only DNA)

pMZ379 only DNA

pMZ379 + sgRNA

Nuclease-free water 43.5 µL 33.5 µL 33.5 µL 33.5 µL

Cut Smart Buffer 5 µL 5 µL 5 µL 5 µL

DNA: 0 µL 10 µL 10 µL 10 µL

SAM: 0.5 µL 0.5 µL 0.5 µL 0.5 µL

CspCI: 1 µL 0 µL 1 µL 1 µL

Total µL 50 50 50 50

8. After adding the enzyme, gently tap the side of the tube to mix the components.

9. Briefly pulse the tubes in your desktop centrifuge in order to collect the components to the bottom of the tubes.

10. Place the tubes in the heat block or water bath set at 37OC for 1 hour and 20 minutes. If using a water bath, leave the tubes in the soft rack.

11. At the completion of the incubation time, place your tubes on ice or store at 4OC until they can be visualized using gel electrophoresis.

Visualizing the DNA Fragments

1. Cast a 0.7% agarose gel with SYBR Safe DNA gel stain using the information from Lab 2 Handout. Store away from light.

2. Keep all DNA on ice.

3. Clean workspace and instruments with ethanol.

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4. Place the five tubes with your restriction digests in a microtube rack.

5. To each tube, add 5 µL of 6X loading buffer. Mix the tubes by gently tapping them. Briefly spin the contents of the tubes down using your desk top centrifuge.

6. Place the agarose gel in the gel box. Align the gel and gel box so that the positive or red electrode will be on the bottom of the box and the negative or black electrode will be at the top.

7. Cover the gel with the appropriate running buffer so that the top of the gel is covered with approximately 5 mm of buffer.

8. Working with your group and the other groups in the lab, load your gels. For each gel, be sure and include 5 µL of ladder in one of the wells.

9. For each sample, add 30 µL of each sample to the appropriate well in the gel. This volume is larger than used before so pipette slowly being sure to stop and hold the pipette at the first stop before removing the tip from the well. Change tips between samples.

10. Place the top on the gel box and plug the electrodes into the top of the box. Match the electrode with the plug to ensure that the proper current has been placed (the black plug with the black electrode and the red plug with the red electrode).

11. Plug the electrodes into the power source. Run at 125V for 20 to 30 minutes.

12. Remove the gel and visualize. The pattern should be similar to that seen in the Figure 2 below.

Figure 2: Restriction Digest of pMZ379 with or without inserted sgRNA.

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Questions 1. What does a restriction endonuclease do?

2. What is a plasmid restriction map?

3. What is the specific purpose of this week’s lab?

4. What is the specific purpose of each control treatment in this procedure?

5. Create a standard curve from your gel, and determine the sizes of your restriction digest products.

6. According to your standard curve, what are the sizes of the DNA in your gel? You

should attach a file or other sheet of paper showing your graph, the best fit line, the equation for the line, the R2 value, and the calculations for determining the sizes of the DNA in your gel. Be sure to label all of the lanes in your gel.

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POST-LAB 1. Be sure all tips and tubes are disposed of in the waste beakers. 2. Wipe down your workstation with ethanol or Lysol. 3. Throw away all gloves and paper towels. 4. Wash your hands. 5. Make sure that all materials are clean and returned to your kit. 6. You will turn in this handout for a grade. Be sure to answer all questions in

the handout. Attach an image of your gel (labeled with the contents of each lane), your standard curve, and all calculations.