Genetics Lab report

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LAB 6: CLONING sgRNA IN A VECTOR PLASMID USING POLYMERASE CHAIN REACTION

OBJECTIVES 1. Generally explain the purpose of a polymerase chain reaction (PCR). 2. Set up a PCR. 3. Explain the role of the chemical components and different temperatures of a PCR. 4. Define gene cloning. 5. Define primer, and explain the importance of primers in PCR. 6. Explain the purpose of using PCR in the context of CRISP/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 following video on PCR: http://www.sumanasinc.com/webcontent/animations/content/pcr.html 3. Complete the pre-lab quiz before lab. LAB SAFETY 1. Wear gloves and goggles 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. GENERAL NOTES ON LAB 1. You will work in groups but will be cloning your own gene. Each student will be

responsible for the PCR of their own gene as well as the back-up of lab partner’s gene. 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 not turn in this lab handout for a grade. Be sure you have all questions answered in the handout, and use the handout as a study guide for the on-line post- lab quiz.

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INTRODUCTION In the previous lab, you were introduced to the process of CRISPR/Cas gene editing, and you familiarized yourself with the workflow that you will follow for the next several labs. (Be sure to review the workflow details from the previous lab’s handout before you continue with this lab.) In addition, you designed the gene for the sgRNA that will serve as a guide for Cas9 to cut the target genes in yeast (Schizosaccharomyces pombe). The DNA gene sequences for the sgRNAs were ordered from a biotechnology company, and will be used in this week’s lab. The sgRNA and the Cas9 genes will be introduced to the yeast cells through a plasmid, called pMZ379. Recall that this plasmid has been designed (by M. Zaratiegui) to have several important features, including:

• a Cas9 gene; • an ampicillin resistance gene; • a nourseothricin resistance gene; • an insertion site for the sgRNA gene, next to a promoter; • several restriction enzyme cleavage sites.

The goal of this lab is to insert the sgRNA gene into the insertion site on the plasmid. This will be accomplished by polymerase chain reaction (PCR), which will not only insert the sgRNA gene into the plasmid, but will also amplify, or create many, many copies of the plasmid. Since many copies of the plasmid will be created, many copies of the sgRNA gene will be created. This process of making many copies of a gene is known as gene cloning. In the next lab, you will visualize the DNA in a gel electrophoresis. INTRODUCTION TO PCR The polymerase chain reaction (PCR) is a technique used to produce large amounts of target DNA. A low concentration of target DNA is incubated with two primers (short stretches of single-stranded DNA) that are complementary to the sequences that flank each end of the target DNA. In this lab, the target DNA is the sgRNA gene and pMZ379, and the primers are the sections that overlap the ends of the insertion site on the plasmid, as shown in Figure 1 below. The new DNA sequences will be copies of each strand of the plasmid, extending from the 3’ end of each primer (see Figure 1.) The essential components of a PCR reaction are:

• the enzyme DNA polymerase (which elongates DNA polymers), • dNTPs (the dinucleoside triphosphates that will be covalently bonded together by

DNA polymerase to synthesize the DNA copies), • a dsDNA template that will be copied, • and single-stranded DNA primers (which define the ends of the DNA that will be

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amplified). In our experiment, the primers are attached to the sgRNA gene to be inserted into the plasmid.

Figure 1. Insertion of the sgRNA gene and primers into pMZ379 by PCR.

The primers serve two purposes. First, the primers provide a free hydroxyl group (-OH) at the 3’ end, onto which DNA polymerase can add new nucleotides to elongate the strand. Second, the primers are designed so that they flank the fragment of interest. The forward primer is found at one end of one strand, and the reverse primer is found at the other end of the other strand. This allows both strands to be elongated in the 5’ to 3’ direction. Example: Suppose you have isolated some dsDNA from a cell. Part of that DNA is shown below. You wish to amplify the segment of dsDNA that is underlined. How could this be done?

3’ …ATC GGG ACC CAC TTT AAA CGT ATC CGT GAC… 5’ 5’ …TAG CCC TGG GTG AAA TTT GCA TAG GCA CTG… 3’

First, you would need to know the sequences at the ends of the fragment (these are known as the flanking ends), in order to create the primers. Once you have identified the sequences at the end of the fragment you want to amplify, you would have a biotechnology company create the primers for your use.

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The primers need to be complementary to each end of the fragment of interest, with one primer for each strand, as shown below.

3’ …ATC GGG ACC CAC TTT AAA CGT ATC CGT GAC… 5’ forward primer 5’ CCC TGG 3’ 3’ ATC CGT 5’ reverse primer

5’ …TAG CCC TGG GTG AAA TTT GCA TAG GCA CTG… 3’ In order to carry out the PCR, all of the components listed above (DNA Pol, dNTPs, primers, buffers, plus the DNA sample containing the fragment to be amplified) are put into a small test tube, shown in Figure 2 below.

Figure 2. Components of a PCR. The PCR tube is then put into a thermal cycler (also thermocycler), which is a machine that can change temperature very rapidly and precisely (see Figure 3.) The thermocycler is programmed to change the temperature of the tubes for precise amounts of time. The machine cycles through the temperatures multiple times, and at the end, many copies of the DNA fragment of interest of are produced.

Figure 3. PCR tubes containing components are placed in a thermocycler.

dNTPs + DNA pol + DNA template + primers

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How PCR Works 1. First, the double-stranded DNA must be denatured. This is accomplished by

heating the DNA to a high temperature (> 90OC). The heat breaks the hydrogen bonds between the bases of the two strands.

2. In a living cell, the enzyme primase creates an RNA primer to begin replication.

Since PCR is done in a test tube, the researcher is in control of what goes into the tube. In PCR, the researcher puts into the tube the two engineered DNA primers, DNA polymerase, some nucleotides, and the template DNA.

3. The tubes are cooled sufficiently (45- 55OC) to allow the primers to anneal

(hydrogen bond) to the single strands of DNA. The process works even if only some of the primers attach to the DNA before the complementary strands reattach.

4. The tubes are then heated to a moderate temperature (72OC), at which the DNA

polymerase works best. At 72OC, DNA polymerase adds the nucleotides to the 3’ end of each primer sequence, complementary to the original strands.

By the end of this first cycle, the following strands have been created (the target

sequence is underlined, and the primers are in bold):

3’ …ATC GGG ACC CAC TTT AAA CGT ATC CGT GAC… 5’ 5’ CCC TGG GTG AAA TTT GCA TAG GCA CTG…..3’

3’….ATC GGG ACC CAC TTT AAA CGT ATC CGT 5’

5’ …TAG CCC TGG GCA AAA TTT GCA TAG GCA CTG… 3’

5. The entire cycle is repeated many times. Eventually, DNA containing the fragment of interest is amplified, or copied many times. The number of strands containing the fragment of interest increases with each cycle. If the cycle is repeated 32 more times, then one original copy of DNA is replicated 232 times. That creates billions of copies of the DNA fragment of interest.

6. In our procedure, the double-stranded circular plasmid will be copied many

times, but the copies will be linear, rather than circular.

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Questions 1. What is the purpose of a polymerase chain reaction? 2. What are the components of a PCR, and what are their general functions? 3. Describe the purpose of exposing the DNA to each of the temperatures during PCR. 4. What are primers, and what is their purpose in a PCR? 5. What is the target DNA in today’s procedure? 6. In a living cell, primase must create an RNA primer to start DNA elongation. Why can DNA polymerase be used in PCR, rather than primase?

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MATERIALS Per Group Quantity Item 1 ea. aliquot 10 μL pMZ379 (kept frozen), 1:100 dilution 4 1.5 mL microfuge tubes and racks 1 ea. 2, 10, 20, 200, and 1000 μL pipettes 1 box ea. sterile pipette tips, large and small 8 0.2 mL PCR tubes 2 foam PCR tube holders 2 foam microfuge tube holders 80 μL PCR Kapa Ready Mix or other appropriate PCR mix 200 μL sterile, nuclease-free water 1 foam cooler for ice 1 bottle ethanol, for disinfecting 1 bottle DNAase Away 1 250 mL beaker for disposal of tips and tubes 1 permanent marker, fine 1 aliquot 100 μL DMSO For the Class Quantity Item 1 ea. forward and reverse primers for target genes, labeled F and R (previously

spun down in centrifuge) 2 thermocyclers (PCR machine) and laptops 1 box ea. latex-free gloves, sm., med., and large safety glasses 1 ice bucket and ice PROCEDURE Preparation 1. Wear gloves and safety eyewear during the procedure.

2. Clean workspace and instruments (pipettors and writing utensils) with ethanol and DNAase away.

3. Obtain a small foam cooler and fill it with ice.

4. Keep all solutions on ice, as instructed, during the procedure. Place the small bottle of nuclease-free water on ice.

5. Obtain one foam microfuge tube holder and two foam PCR tube holders. These will hold your microfuge tubes and PCR tubes in the ice while you work.

6. Make sure that you have all of the materials listed above before you begin.

7. Don’t forget to use a new micropipette tip for each transfer of a liquid.

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Creating the Primer Mix for PCR 1. Obtain two sterile microfuge tubes, one for each gene being modified. You will be

responsible for your own gene as well as the back-up primers of the gene of one of your lab partners. Label the two tubes as ‘A’ and ‘B’. You will make a primer mix in these tubes that contains the forward and reverse primers, and water. Write down the gene that corresponds to each set of primers that you are assigned:

Tube ‘A’ are for gene: _____________________

Tube ‘B’ are for gene: _____________________

2. Your instructor will provide the primers designed in the previous lab. Be sure to identify which tube contains the forward (F) and which contains the reverse primer (R) for each gene before continuing.

3. Place the two tubes in the microfuge holder on ice. To your first microfuge tube (e.g., Tube A), add each of the following:

a. 2 µL reconstituted forward primer; b. 2 µL reconstituted reverse primer; c. 16 µL sterile, ice-cold, nuclease-free water.

4. Repeat Step 3 with the second tube and appropriate primers for the second gene.

5. Keep these primer mixes on ice until needed in a later step.

Preparing PCR Mixes 1. Obtain six small PCR tubes.

2. Label both the sides and tops of six sterile PCR tubes with the letters for your primers. For example:

A1, A2, A3, B1, B2, B3

3. Place the tubes in a white foam tube rack and place them on ice. As you add reagents to each tube, you will move it to the second foam tube rack. This step is crucial for helping you remember which tubes have already received a reagent.

Keep all materials on ice. Begin with the A tubes, and add the liquids as instructed in Step 4. When you are finished with the A tubes, repeat the process with the B tubes. If you do them at the same time, you will likely get confused and mix up the primers.

The experimental treatments will have the following concentrations of solutions.

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Final Concentration

Volume per reaction (25 µL)

pM2379 DNA (1:100 or 1:1000)

1 ng (40fg/µL) 1 µL

Primer mix (10 µL /primer) 0.4 nM/primer 1 µL Kapa Ready mix PCR (2x) (Phusion mix)

1 x 12.5 µL

DMSO (100%) 2 % 0.5 µL Sterile deionized water 10 µL

Table 1. Final concentrations of solutions in experimental treatments

4. Add the materials to each tube indicated in the chart below, as follows: • Make sure that your pipettor is set to the correct volume before you transfer a

liquid. • Always use a new pipette tip for the transfer of every liquid. • After you add materials to a tube, move it from one tube rack to the other, and

have your lab partner check it off on the chart. • To begin, add the materials in the first row of the chart, in the amounts

indicated, to each tube. • Next, add the materials in the second row of the chart to each tube. • Continue adding materials to the tubes, row by row, using clean pipette tips, and

moving the tubes back and forth between tube racks. • When you are finished, all of the tubes should have the same amount of liquid in

them.

Component

Tube 1 (1:00 plasmid)

Tube 2 (no plasmid)

Tube 3 (no primers)

pMZ379 DNA (1:100 dilution) 1 µL 0 µL 1 µL

Primer mix (10 µL /primer) 1 µL 1 µL 0 µL

Kapa Ready mix PCR (2x) 12.5 µL 12.5 µL

12.5 µL

DMSO (100%) 0.5 µL 0.5 µL 0.5 µL

Sterile nuclease-free water 10 µL 11 µL 11 µL

TOTAL 25 µL 25 µL 25 µL

Table 2. Volumes of solutions for PCR.

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5. Press firmly down on the tops of the tube, to make sure they are closed tightly. 6. Repeat Steps 3 – 5 for the second primer set (Tube B).

7. Flick each of the tubes to ensure the components are mixed. Spin down the

contents with a 2 second spin in a desk top centrifuge. Running the Thermal Cycler 1. When all of your tubes are filled, mixed and spun down, load the tubes with tabs

facing the same direction into the PCR thermocycler, and press firmly down on tops of tubes to ensure they are at an even height with one another.

2. Once the thermocycler is filled, perform the PCR according to following protocol:

Number of cycles Temperature Duration 1 98°C 2 min 25 98°C 10 sec 55°C 30 sec 72°C 5 min 30 sec 1 72°C 5 min

Table 3. PCR Protocol for cloning sgRNA plasmid. 3. When the PCR is finished, the tubes should be stored at 4OC until the next lab.

NOTE: PCR products may be stored at -20OC for longer periods.

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Questions 1. What is the specific purpose of each tube in the above PCR protocol? 2. Now is the time to start identifying errors and potential errors in your experiment.

Describe at least two potential errors that may have occurred at this step in the workflow.

POST-LAB 1. Be sure that 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. Empty coolers of ice.

5. Make sure that all materials are clean and returned to your kit.

6. You will not turn in this handout for a grade. Instead, be sure you have all questions answered in the handout, so that you can use it as a study guide for the on-line post- lab quiz.

REFERENCES Mikel Zaratiegui (Addgene plasmid # 74215; http://n2t.net/addgene:74215;

RRID:Addgene_74215).