DNA Analysis- PCR PDF

Chapter 2 – DNA Analysis- PCR Chapter 2 Analyzing DNA by PCR Summary: In the previous chapter we discussed the construction and utilization of cDNA libraries to analyze genes from an organism. In this chapter we will discuss how to determine the s...

Chapter 2 – DNA Analysis- PCR Chapter 2 Analyzing DNA by PCR Summary: In the previous chapter we discussed the construction and utilization of cDNA libraries to analyze genes from an organism. In this chapter we will discuss how to determine the size of the cDNA insert on a plasmid using PCR and agarose gel electrophoresis. The first part of this chapter describes the process of PCR and how to set up a reaction to amplify a DNA fragment. In the second part we will discuss the process of gel electrophoresis and how to analyze an agarose gel that contains PCR samples. I. Amplifying DNA by Polymerase Chain Reaction (PCR) A. Introduction Sequencing or cloning reactions to analyze DNA often requires relatively large quantities of DNA. However, many interesting fragments of DNA are often found in nature in extremely small amounts. This is due in large part to the fact that the genome is incredibly long. Once genomic DNA is isolated, a specific fragment of DNA (e.g., gene) only comprises a miniscule fraction of the total. To make matters worse, genomic DNA is often difficult to isolate in large amounts. The Polymerase Chain Reaction (PCR) overcomes these technical difficulties. PCR is a form of DNA replication where a specific region of a DNA sample is selectively amplified in a solution with other DNA sequences. This amplification process is repeated multiple times, resulting in over a billion-fold amplification of a specific DNA fragment. PCR has several constituents that are required in any replication reaction: i) DNA template: This is a DNA sample that contains the specific region that you want amplified. Since PCR is a synthesis process in which a DNA fragment is amplified more than a billion times, only a very small amount of template is needed. ii) Taq Polymerase: Taq is a heat-stable DNA polymerase enzyme that synthesizes the DNA using the sample DNA as a template. iii) Oligonucleotide Primers: The reaction requires short oligonucleotides that serve as primers for replication. Like any DNA polymerase, Taq Polymerase cannot synthesize DNA on a naked single stranded DNA molecule (cannot perform de novo synthesis), but can only extend from a primer. Two specific oligonucleotide primers that anneal on either side of the region to be amplified are used in the WSSP project. iv) dNTPs: Deoxyribonucleotide triphosphates are the building blocks (monomers) that the Taq polymerase uses to synthesize the new DNA (a polymer). B. Steps in PCR: There are several steps in the DNA synthesis process that are carried out for multiple cycles in a PCR. These steps require the sample to be incubated at specific temperatures for short periods of time. PCRs are therefore usually carried out in the thermal cycler, a device that holds a ã 2022 WSSP 2-1  Chapter 2 – DNA Analysis- PCR temperature block that can be programmed to rapidly change temperatures. The three steps in each cycle of the PCR are listed below (Fig 2-1). i) Denaturation Step: The first step in the WSSP PCR is to denature the DNA sample that will be amplified. The sample DNA is placed in a microcentrifuge tube and heated for about 1 minute at 95˚C. This heating disrupts the hydrogen bonds holding the strands of the double helix together, and the double–stranded DNA denatures into single–stranded DNA. ii) Primer Annealing Step: After denaturation at 95˚C for 1 minute, the thermal cycler quickly drops the temperature of the reaction to 50˚C and maintains this new temperature for another 2 minutes. Under these conditions, the two oligonucleotide primers (P1 and P2 in Fig 2- 1) anneal (base pair) to their complementary sequences on either side of the region to be amplified on the denatured genomic DNA. The primers in a PCR reaction are usually 15- 25 nucleotides long. They are highly specific for the region we are trying to amplify and should not anneal to other genomic sequences. These primers would be synthesized chemically using an oligonucleotide Fig 2-1. Steps in a single cycle of PCR using the synthesizer, which can synthesize any DNA P1 and P2 oligonucleotide primers (green) to amplify the DNA coding for the gene (red box). sequence that you want up to about 100 nucleotides long. Thus, our PCR would Blue indicates the newly synthesized DNA. contain two primers: one complementary to the DNA on one end (P1) and the other 1N complementary to the DNA on the other end Cycle 1 (P2). 2N iii) Synthesis Step Cycle 2 In the final step of the PCR reaction, the 4N temperature is raised to 72˚C for 2 minutes (Fig 2-1). This allows Taq Polymerase to bind Cycle 3 to the 3’ end of the primers and synthesize the complementary strand of DNA using the genomic DNA as a template. Note that the 3' 8N ends of both primers "point" towards the region that is going to be amplified. Since all DNA polymerases synthesize DNA in the 5' to 30 Cycles 3' direction, this means that the DNA between 1 X 109 N the primers is replicated. Fig 2-2. Multiple cycles of PCR geometrically amplify the DNA fragment. ã 2022 WSSP 2-2  Chapter 2 – DNA Analysis- PCR The three steps described above generate two copies of the target region from the original one. The denaturation-annealing-synthesis steps comprise one cycle. By repeating this cycle 25-30 times, it is possible to obtain enormous levels of amplification (Fig 2-2). Theoretically, after ten cycles, a single copy of the gene has been amplified into 1024 copies; after twenty cycles, it has been amplified into 1,048,576 copies; and after thirty cycles, 1x109 copies. 30 cycles take about two to 3 hours to complete. A video of this process can be downloaded at: http://www.dnalc.org/resources/animations/pcr.html. The complete PCR cycle is: I. Initial Denaturation 94˚C for 5 minutes (This insures that the DNA is thoroughly denatured.) II. Amplification (The PCR thermal cycler will repeat these three steps in order 30 times.) 94˚C for 1 minute (Denaturation of target DNA) 50˚C for 1 minute (Annealing of primer to template DNA) 72˚C for 1 minute (Elongation of primer to produce new DNA strand) III. Additional Elongation (This step insures all DNA strands are full length) 72˚C for 5 minutes IV. End Program After step III, the PCR machine has been programmed to drop the temperature of the heating block to 4°C. Your samples will be very stable if left at 4°C. C. Taq Polymerase If you have been paying close attention, you should have noticed a problem. A typical PCR cycle goes from 94˚C (denaturation) to 50˚C (annealing) to 72˚C (synthesis). And yet, enzymes should be kept cold to maintain stability! Why doesn't the PCR reaction stop at the first 95˚C temperature? The reason is that Taq Polymerase is an unusual enzyme. This DNA polymerase is purified from a bacterium that grows in hot springs. Thus, Taq polymerase (and Vent polymerase obtained from a bacterium in deep ocean thermal vents) are unusually stable. In fact, the 72˚C (163˚F) temperature for extension is the optimal temperature for Taq polymerase to synthesize DNA. ã 2022 WSSP 2-3  Chapter 2 – DNA Analysis- PCR II. Determining the size of your clone insert using PCR A. Using PCR to determine the size of the insert. We can use PCR as a method to determine the size of the DNA insert in a plasmid. We will use two primers, called Forward (For) and Reverse (Rev), that hybridize on either side of MCS cloning site (Fig 2-3). These primers will amplify only the DNA in between the primers. The fragment generated would be the size Fig 2-3. PCR amplification of the cDNA insert in of the insert plus the size of the vector pTriplEX2 region on either side of the insert from the position where the primers hybridized to the SfiI sites where the insert was cloned. As a result, the PCR fragment is going to be a little larger (roughly 200 bp) than the DNA insert. B. Using bacterial cultures for the plasmid DNA template: We could perform the PCR using plasmid DNA that we purified from the bacteria (as discussed in Chapter 1). However, since PCR only needs a very small amount of template DNA to amplify a specific fragment we actually do not need to purify the plasmid DNA. Instead we can directly take some of the bacterial ON culture and add a few of the cells to the PCR mix. During the first step of the PCR cycle, in which the sample is heated to 94°C for 5 minutes, the bacteria will break open, and the plasmid DNA will denature. Even though there is a large amount of the bacterial chromosomal DNA in the reaction tube, the oligonucleotide primers will only hybridize to complementary sequences on the plasmid. Therefore, only the insert fragment on the plasmid will be amplified. C. Setting up a single PCR sample: To perform PCR, you will need template DNA (contained on the plasmid from the bacterial ON culture) and oligonucleotide primers (For and Rev). However, you will also need the Taq Polymerase enzyme, the deoxy-nucleotides (dATP, dGTP, dCTP, dTTP) and the proper buffers to carry out the reaction. In many laboratories each of these components are added individually to the reaction tube. However, in the WSSP we are fortunate to be able to use a “2X Taq Mix”, which contains Taq Polymerase enzyme, nucleotides and the proper buffers to carry out the reaction. Therefore, to set up a reaction with the mix all you will have to do is add some water, the For and Rev nucleotide primers, and a small aliquot of your bacterial ON culture. Below is the protocol for setting up a single reaction with a 2X Taq Mix. (NOTE: If you are using GE Taq Beads for your PCR please follow the procedure in the laboratory manual. Although the volumes and reagents are different than those used with the 2X Taq Mix the concepts are the same). 1. Dilute the ON bacterial culture 40-fold by adding 5 µl of the overnight culture to an appropriately labeled microfuge tube containing 200 µl of dH2O (distilled water). Mix each tube by vortexing or tapping the tube. ã 2022 WSSP 2-4  Chapter 2 – DNA Analysis- PCR As depicted on the Jurassic Park movies and on CSI television shows, PCR requires only a small amount of template DNA to amplify a DNA fragment. In fact, too much template DNA is actually a bad thing! This is because all the primers and nucleotides will be used in the first few cycles of the reaction. As a result, there will be less amplification of the specific fragment. Due to the high number of copies of the plasmid in each cell and large number of cells in a small volume, if you add undiluted ON bacterial culture to the PCR, it will not work. We therefore only need to add a small amount of the ON culture. NOTE: These diluted samples can be placed in the freezer after this step and the setup of the PCR can be completed another day after the samples are thawed and mixed. DO NOT DISCARD THE CULTURES!!! You will need these ON cultures for the plasmid DNA minipreps in a later step if you find that the clones have large inserts. We recommend that you centrifuge the ON culture in a microfuge tube, discard the supernatant, and then resuspend the bacterial pellet in Buffer P1 of the miniprep kit (See the plasmid purification procedure in Chapter 1B). The resuspended cells can then be stored in the freezer for weeks until you are ready to continue the steps with miniprep purification. 2. If you are using a 2X Taq Mix: Add the following reagents directly to the PCR tube in the following order: 1 Rxn. Sterile double distilled (dd)H2O 7.5 µl For Primer (10 pmole/µl) 2.5 µl Rev Primer (10 pmole/µl) 2.5 µl 2X Taq Mix 12.5 µl Diluted cells from Step 1 2 µl =25 µl 3. Add your tubes to the thermal cycler machine and start the program. The complete amplification takes about 2.5 hours. I. Initial Denaturation 94˚C for 5 minutes (This insures that the cells are lysed and the DNA is thoroughly denatured.) II. Amplification (The PCR thermal cycler will repeat these three steps in order 30 times.) 94˚C for 1 minute (Denaturation of target DNA) 50˚C for 1 minute (Annealing of primer to template DNA) 72˚C for 1 minute (Elongation of primer to produce new DNA strand) III. Additional Elongation (This step insures all DNA strands are full length) 72˚C for 5 minutes IV. End Program After step III, the thermal cycler has been programmed to drop the temperature of the heating block to 4°C. Your samples will be very stable if left at 4°C. 4. Store the samples in the freezer until you are ready to run your agarose gel. ã 2022 WSSP 2-5  Chapter 2 – DNA Analysis- PCR Although it is unlikely there are any enzymes in the reaction that will degrade your DNA, it is always best to store DNA samples in the freezer to reduce the probability that they will be degraded. D. Setting up PCR for multiple samples When setting up PCR for several ON cultures, it is very useful to make a “master mix” containing all ingredients except the DNA. Then you can aliquot the appropriate volume of master mix to separate tubes, followed by the addition of the appropriate diluted bacterial ON culture to each tube. Preparing such a “master mix” will help avoid a lot of repetitive pipetting which takes time and can result in errors. The recipes for the reaction mixtures are given below. Notice that there will be more mix than you will need (one more than the number of reactions being performed, thus allowing for pipetting Fig 2-4 Set up a Master errors so you don’t run out of mix. mix and aliquot for multiple PCRs. 1. Using 2X Taq Mix to set up the PCR: a. As described in the steps for a single PCR, the first step is to dilute each of the bacterial ON cultures 40-fold by adding 5 µl of the overnight culture and to the appropriately labeled microfuge tube containing 200 µl of dH2O. Mix each tube by vortexing or tapping the tube. If students are working in groups, each student should perform this dilution on their cell sample. b. If you are setting up more than two PCR samples you should make a PCR mix to aliquot into each of the reaction tubes. Prepare a mix for one more sample than the number of samples that you will actually be running (i.e. if you are going to run 3 samples, prepare the 4X mix, for 4 samples prepare the 5X mix). We recommend that you work in groups of 4 students and perform the experiments on one clone per student at a time. Thus, in this case the group should prepare a 5X mix for 4 clones. i) In this tube prepare a reaction digestion mixture as shown as shown below. 1X 3X 4X 5X sterile ddH2O 5.5 µl 16.5 µl 22 µl 27.5 µl For Primer (10 pmole/µl) 2.5 µl 7.5 µl 10 µl 12.5 µl Rev Primer (10 pmole/µl) 2.5 µl 7.5 µl 10 µl 12.5 µl 2X Taq Mix 12.5 µl 37.5 µl 50 µl 62.5 µl Diluted Cells ** (DO NOT ADD THE BACTERIA!) ** Mix the ingredients of the “PCR Mix” by gently tapping the tube. c. Label the sides of the PCR tubes with the clone names. d. Use a P200 to add 23 µl of the “#XMix” to each PCR tube. ã 2022 WSSP 2-6  Chapter 2 – DNA Analysis- PCR e. Add 2 µl of the appropriate diluted colony that was prepared in Step 1a (above). Mix by gently tapping each tube. f. Set the tubes in the thermal cycler and start the program as described above. III. Agarose Gel Electrophoresis: See videos on pouring and running an agarose gel. A. Introduction: Once we have performed the PCR on the plasmid clone, the next step is to determine the size of the amplified DNA insert. To do this we want to separate the different fragments on the basis of their size (length). Gel electrophoresis is the standard method used to separate, identify and purify DNA fragments. The phosphate groups on the DNA backbone confer a net negative charge on the molecule. Thus, if a solution of DNA is placed into an electric field, the DNA Fig. 2-5 In gel molecules will migrate toward the positively charged electrode. In gel electrophoresis DNA electrophoresis, the DNA fragments are forced to move through a porous strands (red) must snake matrix consisting of agarose (Fig 2-5). The gel can be thought of as a through the gel matrix. maze of agarose and open spaces. When DNA moves through the gel, it Smaller fragments do this must “snake” around the agarose, fitting through the pores. Large better than larger fragments fragments of DNA have more trouble navigating through the gel than do and therefore migrate faster small fragments. Thus, if DNA is placed within a gel and through the gel. electrophoresed for a limited time, fragments will separate according to their sizes, with smaller fragments migrating further from the origin than larger ones. B. Steps in running an agarose gel 1. Pour agarose gel: In the WSSP we use agarose slab gels to separate the DNA fragments. Agarose is melted in a buffer and then allowed to cool in a casting tray to form a jello-like consistency. The casting tray contains a plastic comb that creates indentations (wells) in the Fig. 2-6 (Left) Diagram of DNA (blue) being loaded into an agarose slab gel. (Right) DNA migrates through the gel once a current is applied to the gel. Dyes are used to monitor the progress of the electrophoresis. ã 2022 WSSP 2-7  Chapter 2 – DNA Analysis- PCR agarose when it is removed (Fig 2-6). The hardened gel is then placed in a gel box with electrodes and then is covered in buffer. (These gels have been called submarine gels because they are submerged under the buffer). 2. Add gel loading dye to samples: Gel loading dye, which contains EDTA, marker dyes and glycerol, is then added to each of the PCR samples. The addition of EDTA chelates many of divalent cations from the reaction, which inactivates the enzyme. The marker dyes are used to visualize your sample as you are loading your gel. These dyes migrate into the gel during electrophoresis and are useful to monitor how far your DNA has migrated through the gel. The glycerol makes your DNA sample denser than the buffer so that your sample will sink into the gel well and stay there. 3. Load samples and run gel: The DNA samples are loaded into the wells using a pipettor. A current is then applied to the gel and, as described above, the DNA begins to migrate through the gel and separate based on the sizes of the fragments. Linear molecules of double-stranded DNA migrate through gel matrices at rates that are inversely proportional to the log10 of the number of base pairs. Note that if you electrophorese DNA fragments of known size, you could construct a graph that can be used to determine the approximate size of an unknown fragment. As you may predict, the migration rate of a given fragment is dependent on the concentration of agarose used, for example the higher the concentration of agarose the smaller the pores therefore the slower the DNA fragments will migrate. 4. Take a picture of the gel. Stains are used to bind to the DNA fragments and will cause the DNA to shine a fluorescent color when they are exposed to ultra violet (UV) light. These stains allow us to visualize the relative movement of DNA fragments in the gel. Depending on the regulations at your school your agarose gel may contain ethidium bromide or another stain. After the gel has run, you will be able to view the DNA fragments by placing the gel on a UV light box. The DNA fragments will bind the ethidium bromide and the DNA will shine red on the UV light box. You can then take a digital image of the gel. Note that it is best to take a black/white image of the gel instead of color, because it provides better contrast to see the bands. (Note: Ethidium bromide binds DNA and is a known carcinogen. Please wear gloves when handling the gels.) 5. Create a Mock-up version of the gel in PowerPoint or Google Slides: Students are required to take images of all their gels and import these images into PowerPoint or similar “app” (must be able to edit the converted images into “jpg” files) so that these can be properly labeled (mocked up). We have provided a PowerPoint Gel Image template file to use in mocking up your gel (see Fig 2-9). Please see the Lab 4 notes for a detailed description on how the gel should be mocked up. After you have finished mocking up the image, Fig 2-7. Diagram of PCR amplification and gel upload the gel files to your school’s Google electrophoresis of fragment containing the cDNA Docs Clone Report Sheet. insert. ã 2022 WSSP 2-8  Chapter 2 – DNA Analysis- PCR VI. Determining the insert size of a cDNA Library Clone Since we know where the two primers hybridize on the plasmid vector, it is possible to estimate the size of the cDNA insert by measuring the size of the PCR fragment that was generated from the plasmid template (Fig 2-7). Looking at the map of the MCS and where the For and Rev primers hybridize, Fig 2-8 Map of the pTriplEX2 plasmid with the MCS. The SfiI sites a PCR of a plasmid used to clone the cDNA inserts are shown in red. The For and Rev template without an insert primer sites used to determine the size of the inserts are shown in blue. will generate a fragment that is roughly 200 bp (Fig 2-8). All of your PCRs should generate a fragment that was at least this size. Now think about what size the fragment would be if there is an insert in the plasmid. The fragment generated would be the size of the insert (1 Kb in the example) and the lengths on either side of the insert from the position where the primers hybridized to the vector (roughly 0.2 Kb). As a result, the PCR fragment is going to be a little larger (0.2 Kb) than the real length of the insert. We can therefore use this calculation in reverse to determine the size of an insert by measuring the size of the PCR fragment. A. Analyzing Real Data. Below is an example of a gel used to analyze clones from a cDNA library that was constructed by cloning the DNA fragments into the SfiI sites of the pTripleEX2 plasmid (Fig 2-9). Four samples were analyzed by each student and the PCR samples were run on the gel. A commercial 1 kb DNA Marker ladder (M) with DNA fragments Fig. 2-9 A real example of a PCR screen of clones from the cDNA library. M is the 1 KB marker with the sizes of the bands of known sizes was used to shown to the left. (Note: The marker we now use has a 1550 bp determine the size of the inserts. band not 1650) Lane 2 has a PCR of the pTriplEX2 vector without For illustration purposes, a PCR an insert. The rest are PCR of clones from the cDNA library. You of the pTriplEX2 vector without should label your gels like this (mocked up version of the gel) to an insert is also shown (lane 2, post on your Google Docs Clone Report Sheet. labeled pTriplEx2). ã 2022 WSSP 2-9  Chapter 2 – DNA Analysis- PCR Analysis of the gel results should involve the following questions: 1. Did the gel run OK? Look at the gel and determine if there are bands present. The best place to look is the Marker lane (M) because you know there should be DNA there and you should have a rough idea of what it would look like. If bands are present in the expected pattern, then that would indicate the gel was made properly, was electrophoresed in the correct direction, and that the DNA staining worked. If there are no bands present in the M lane or any other lanes then several things could have happened. It is possible the stain did not work, so you cannot visualize the DNA. Alternatively, the leads may have been hooked up backwards, then the DNA would have migrated in the other direction out the top of the gel, which would also result in a blank gel. Finally, it is also possible that you forgot to add the DNA to your samples so that all the lanes were blank. If the gel or running buffer were improperly made, then that would cause the DNA bands to run abnormally. This can also happen if the gel is run too fast or with too high a voltage. The smeary gel in Fig. 2-10 shows a minor Fig 2-10. An example of a example of this type of problem. Streaks that interrupt the poor gel. The streaks are bands are likely caused by chunks of agarose that were not likely from incomplete completely melted. Since these chunks contain a very high melting of the agarose. percentage agarose, the DNA cannot migrate through them. In Fig 2-9, the markers are well defined and look as expected. Even though the bands are not as clear as you would like the data is still valuable. By looking at the pattern it is possible to determine the size of each band (Fig 2-10). This will be very useful when you need to determine the size of the bands in the digests and in the PCR of your clones. You will need to mark these sizes on the pictures of your gel. 2. Did the PCR work? Is there a band present in the PCR lane? If there is, then the reaction worked. If there is no band present, there are several possible explanations. i. One is that you did not run the reaction properly, i.e. forgot to add a primers or the bacterial ON culture. Keep in mind, however, that in setting up the PCR, you made a mix of the primers. Therefore, if the PCR reactions worked for your other clones then you can rule out that it was a problem with adding primers or with the thermal cycler. However, you may still have forgotten to add the ON culture. Fig 2-11 New 1KB Plus ii. If all your reactions did not work, it could be that your thermal marker sizes Note the cycler was not properly programmed. Check the program to be sure band is now at 1500 instead of 1650 that it has the correct temperatures and times for each step. ã 2022 WSSP 2-10  Chapter 2 – DNA Analysis- PCR iii. Another possibility is that one of the primer hybridization sites in the plasmid was deleted in the cloning process. Sometimes during the digestion of the vector or ligation of the insert a portion of the vector is deleted. If this region includes one of the primer hybridization sites, then you will not get proper amplification. However, this is a rare event and so it is likely to occur only in one sample. If the other lanes also do not have a band then it is probably a different problem. In addition to the above possibilities, many additional factors could affect the success of the DNA agarose gels that should be explored and discussed. For example, many weak bands could indicate primers not hybridizing specifically, a smear could indicate too much template DNA was used, etc. 3. What size is the insert? Estimate the size of the insert by comparing the migration of the PCR band with the different marker fragments. Since the size of the PCR band includes the vector regions on either side of the insert, you will need to subtract the length of these regions (200 bp) from the size of the PCR fragment to determine the correct size of the insert. Below are examples of the analysis of the first few lanes. pTriplEX2 (Fig 2-9, Lane 2): The estimated size of the PCR in this lane is 200 bp. This agrees with the predicted size of a PCR from a vector without an insert. If you find fragments in this size range it is likely there is no insert or a very small (less than 50 bp) insert. This sample would not be good for further analysis. 20JM1.10 (Lane 3): In this lane the PCR fragment is roughly 600 bp long. Since 200 bp of this fragment is the vector region that is amplified in the PCR, then this indicates the estimated size of the cDNA insert is 400 bp. This clone should be further analyzed. 20JM2.10 (Lane 4): In this lane the PCR fragment is roughly 1700 bp long. Since 200 bp of this fragment is the vector region that is amplified in the PCR, then this indicates the estimated size of the cDNA insert is 1500 bp. This clone should be further analyzed. 20JM3.10 (Lane 5): The dotted line in the gel is at the 500 bp size. The band in lane 5 is just below this line and therefore the estimated size of the PCR fragment is roughly 450 bp long. Since 200 bp of this fragment is the vector region on either side of the insert that is amplified in the PCR, then this indicates the estimated size of the cDNA insert is 250 bp. Since this is less than the 300 bp that are required for submission of the DNA sequence to NCBI, this sample would not be good for further analysis. The rest of the clones can be analyzed in the same manner. 4. Record your predictions. It is very important to record your conclusions from your analysis of the gel. If you do not and then return to analyze the gel at a later date you may arrive at different conclusions. a. Be sure to enter the predicted sizes of the insert on the mock-up of the gel (See Lab 4 for details on mocking up a gel image and posting it on Google Docs). b. Enter the predicted sizes of the inserts on the Google-Docs Clone Report sheet. ã 2022 WSSP 2-11  Chapter 2 – DNA Analysis- PCR 5. If a clone is to be further analyzed, the next step is to perform a miniprep on the plasmid DNA. See the miniprep procedure in Chapter 1B of the lecture notes for more information. ã 2022 WSSP 2-12