BIO605 Exam Review - Bacterial Transformation - PDF

BIO605 EXAM REVIEW WEEK 1 – Bacterial Transformation Bacterial transformation is a process by which bacteria take up foreign genetic material (such as plasmids) from their environment and incorporate it into their own genome. Competence: Competence refers to the ability of bacteria to take up extrac...

BIO605 EXAM REVIEW WEEK 1 – Bacterial Transformation Bacterial transformation is a process by which bacteria take up foreign genetic material (such as plasmids) from their environment and incorporate it into their own genome. Competence: Competence refers to the ability of bacteria to take up extracellular DNA from their surroundings and incorporate it into their genome. 1) Name two ways in which bacteria can be made competent in the lab. Chemical transformation: bacteria are treated with certain chemicals (such as calcium chloride) that disrupt the cell membrane, making it permeable to DNA uptake. Electroporation: Bacteria are briefly exposed to an electric field, which creates temporary pores in the cell membrane, allowing DNA to enter 2) Describe the role of calcium ions in making cells chemically competent. Calcium ions stabilize the negtaivly charged DNA and the negatively charged phospholipids inm the cell membrane. Tis disruption allows DNA molecuels present in the surrounding medium to enter the cell. 3) Explain what happens to the cell wall during electroporation. During electroporation the electric field causes temporary pores to form in the bacterial cell membrane. This disruption allows DNA molecules present in the surrounding medium to enter the cell. 4) Explain why we incubate cells in the absence of antibiotic prior to plating. Prior to plating incubating cells in the absence of antibiotic allows for recovery and expression of the newly acquired genes without selective pressure. This ensures that all transformed cells have equal opportunity to express the antibiotic resistance gene carried on he plasmid. 5) Explain the role of each of the following features of a plasmid cloning vector: origin of replication, multiple cloning site (sometimes called the polylinker), selectable marker. Origin of replication: this specific DNA sequence where replication of the plasmid begins. It ensures that the plasmid is replicated along with the bacterial genome during cell division. Multiple cloning site (polylinker): this is a region on the plasmid that contains multiple restriction enzyme recognition sites allowing for the insertion of foreign DNA fragments. Selectable marker: This is a gene often encoding antibiotic resistant, which allows for the selection of bacterial cells that have successfully taken up the plasmid. 6) Explain which feature of the pUC19 plasmid cloning vector makes it a “high copy” plasmid. The pUC19 plasmid cloning vector contains a high copy number of origin of replication. This means that once inside the bacterial cell, the plasmid is replicated many times, resulting in a high number of copies per cell. 7) Propose a way to select for bacterial cells that have successfully been transformed with pUC19. Bacterial cells transformed with pUC19 can be selected by plating them on agar plates containing an antibiotic (such as ampicillin) to which the plasmid confers resistance. Only transformed cells containing the pUC19 plasmid will survive on the antibiotic-containing plates, allowing for their selection and subsequent analysis. WEEK 2 1) What is the “purpose” of the PCR, and why is it a useful laboratory technique? The purpose of a PCR is to pick a piece of DNA and amplify that specific DNA fragment from a larger segment of DNA. It is a very useful laboratory techniques because of its role in amplifying DNA fragments in a very rapid manner producing millions to billions of copies of that specific DNA fragment. 2) List the ingredients that must be included in any PCR, and in a sentence or two explain what each one does. - Template DNA – The starting material for the PCR. It is a sample of DNA that contains the target sequence that is going to be amplified. DNA polymerase – typically Taq polymerase, catalyzes he synthesis of new DNA strands by adding the nucleotides onto the primers. Primers – The starting point for DNA synthesis, consisting of short singlestranded DNA oligonucleotides the anneal to the complementary sequences next to the target region. Nucleotides – required for DNA synthesis. - Thermal cycler – PCR machine used to cycle through a series of temperature ranges. Buffers – creates optimal conditions for the PCR to run smoothly. 3) What are the key steps in a PCR thermocycling program, and what happens during each? Denaturation – The machine raises its temperature above the DNAs melting point which separates the DNA strands. Annealing – The primers bind to the target DNA fragments by lowering the temperature of the thermal cycler. Extension – The temperature rises to aloe for the DNA pol to extend the primers by adding nucleotides to the growing DNA. 4) Define Tm in terms that make sense to you. You may need to Google Tm or melting temperature for this because it isn’t explicitly described in the readings. Tm is the temperature at which half of the oligonucleotide molecules are single stranded whole the other half remain double stranded. 5) Why/how is Tm relevant to PCR? Tm plays an important role in PCR because it can help you prep before starting a PCR reaction because it is important to have primers that are around the same Tm, so they anneal and dissociate from the DNA at the same temperatures. 6) What do you think would happen if you performed a PCR using an annealing temperature that was significantly (say, 10 degrees Celsius) higher than the Tm for the primers? (Assume that the two primers used in the reaction have similar Tm.) What do you think would happen if you performed a PCR using an annealing temperature that was significantly lower than the Tm for the primers? From what I know about Tm and PCR reaction I would think that selecting an annealing temperature that is significantly higher or lower that the Tm for primers can lead to many consequences like, efficiency, decreased nonspecific amplification and primer-dimers could form. 7) Draw a quick sketch or describe in words a primer hairpin and a primer dimer. Does your sketch help you understand why they are called homoduplexes and heteroduplexes, respectively? Primer dimer: When the primers anneal to each other rather than the DNA segment and a short double-stranded DNA fragment is formed which can reduce the efficiency of the overall PCR reaction. Primer hairpin: When the primer fold back on itself and hairpin structure forms which can cause the primer to not properly anneal to the template DNA. 8) In a sentence or two, describe the main difference between the Taq and Phusion polymerases. What is meant by the term “fidelity” when used to describe enzyme activity? The main difference between Taq polymerase and Phusion polymerase is that the Taq polymerase lacks the proofreading activity which can cause more errors during DNA synthesis while Phusion polymerase does have those proofreading capabilities. The term fidelity refers to the accuracy with which an enzyme synthesizes or replicates a nucleic acid sequence without introducing errors or mutations. 9) Given that we are eventually going to clone our PCR products in a vector suitable for producing protein in vivo, which polymerase do you think we should use, and why? I think that we should use the Phusion polymerase for our PCR reactions because I offers higher fidelity and lower error and mutation rates then Taq polymerase does. 10) Calculate the volume of each stock reagent necessary for a 50 μL reaction, and add it to the table. 11) Summarize two advantages of using the Phusion Hot Flex Master Mix. The two big advantages to using Phusion Hot Flex Master Mix is that the master mix includes buffer, enzyme, and dNTPs, reducing preparation time and pipetting errors and the "hot start" feature help to ensure enzyme inactivity at room temperature, which only is activated after the initial denaturation step. 12) Why is it important to include a negative control when setting up PCRs? Overall, it is important to include a negative control in PCR experiments because it can serve as a quality control tool to help detect any potential sources of error with any of the ingredients in a PCR which will increase the overall accuracy. 13) Why is it important to include a positive control when setting up PCRs? Why might one choose not to include a positive control for a primer:template combination that has been optimized? Positive controls can help to ensure the reliability and accuracy of the PCR reactions, but some may choose not to use a positive control in their experiments because some primer: template combinations have already been validated and they have been seen. through thoroughly which could allow for researchers to just skip the step in an effort to save time and resources. 14) What are possible ways to shrink the differnec in Tms between the forward and reverse reaction Some possible ways to shrink the difference in Tms between the forward and reverse primers could be adjusting the primer length or add or remove nucleotides from the sequence. 15) In a few sentences, summarize what you learned about the Gateway cloning system from your reading. What are a few of the advantages of the system? What questions do you still have? From the reading, I learned that the Gateway cloning system offers a fast, efficient, and versatile method for cloning DNA fragments. It utilizes att recombination sites and specific enzyme mixes to enable rapid and highly efficient cloning without the need for overnight incubations. The system allows for the cloning of various types of DNA fragments, including PCR fragments, cDNA, and genomic DNA, and it is applicable to a wide range of organisms. The system involves the creation of entry clones containing the gene of interest flanked by attL recombination sites, followed by LR or BP reactions to generate expression clones or new entry clones, respectively. I am still wondering what the limitations to this system are and how widely can it be used? 16) Why will the PCR primers we use next week contain Gateway adapter sequences? We will be using PCR primers from the Gateway adapter sequence because it takes advantage of site-specific recombination. 17) Open and skim through the Drosophila cDNA sequences on Blackboard. The coding sequence for the genes is written in capital letters. Notice that there are no introns. Explain why that is. Hint: you might need to remind yourself what a cDNA is to answer this question. cDNA is synthesized from mature mRNA molecules through the process of reverse transcription. Unlike DNA, which has both exons and introns mRNA molecules have already undergone splicing to remove introns and retain only the exons. Therefore, when cDNA is synthesized from mRNA, it reflects the mature, spliced mRNA sequence and contains only the exon regions corresponding to the protein-coding sequences. Week 3 18) Explain why gel electrophoresis is a useful technique in molecular biology. Gel electrophoresis is a useful technique in molecular biology because it allows you to separate DNA, RNA, and many proteins based on their size. 19) Describe the basic principles underlying how gel electrophoresis “works”. The process of gel electrophoresis includes employing a gel matrix, typically agarose. The solution is loaded into the wells of the gel and then the nucleic acid molecules are separated by size using an electric field, with negatively charged molecules migrating towards the positive pole. The migration rate is determined by the molecular weight, resulting in smaller molecules moving faster than larger ones. 20) Explain the role of buffers in gel electrophoresis. Buffers in gel electrophoresis help to maintain a astable pH, facilitate an even electrical conductivity, stabilize charged molecules. 21) Describe the relationship between DNA charge, size, and shape and electrophoretic mobility. DNA molecules with a negative charge migrate towards the positive electrode. The smaller DNA fragments move faster than larger ones because they experience less frictional resistance in the gel. The shape of the DNA molecules can also impact their mobility because the longer and skinny the molecule the more predictably it will travel then big circular ones. 22) Describe how the gel matrix (agarose vs. polyacrylamide) and the concentration (%) of gel used affects the resolving power of the gel. The agarose gels may be preferred for separating the larger pieces of DNA because of its larger pore sizes while the polyacrylamide has smaller poor size which give a higher resolution for smaller DNA fragments. If you increase the concentration in agarose gel the pores shrink and if you increase the concentration of polyacrylamide gel, then the pores shrink even more. 23) Explain why it is important to run DNA markers (sometimes called "ladders") along with experimental samples. It is important to run DNA markers along with the experimental samples because it ensures accurate size determination, quality control, standardization, and quantification of DNA fragments. 24) Explain how nucleic acids are visualized after gel electrophoresis. Nucleic acids are visualized after gel electrophoresis using various staining techniques like fluorescent dyes, then after the gel is run, they are seen visible under a UV light. 25) Outline the steps one takes to "extract" nucleic acids from a gel using the QIAquick gel extraction kit. Here's a simplified breakdown of the procedure: 1. Excise Gel Fragment: Cut out the DNA fragment from the gel using a clean scalpel. 2. Prepare Gel Slice: Remove excess agarose to minimize the size of the gel slice. 3. Weigh and Dissolve: Weigh the gel slice and dissolve it in Buffer QG. Incubate at 50°C until completely dissolved. 4. Adjust pH: Ensure the mixture turns yellow. If not, add sodium acetate to adjust the pH. 5. Precipitate DNA: Add isopropanol to the sample to precipitate DNA fragments. 6. Bind DNA: Place a QIAquick spin column in a collection tube and apply the sample to the column. Centrifuge to bind DNA. 7. Wash: Wash the column with Buffer PE to remove agarose and residual contaminants. 8. Elute DNA: Add Buffer EB to elute purified DNA from the column. 9. Prepare for Analysis: If needed, mix purified DNA with Loading Dye before loading onto a gel for analysis. 26) Explain the relationship between pH and salt concentration and DNA binding to the QIAquick column. The appropriate combination of pH and salt concentrations create optimal conditions for the DNA to bind to the QIAquick column. If the pH values are out of the appropriate range it can affect the charge on the DNA and similarly if there is too much or too little salt the DNA may not bind properly. 27) Briefly describe why one might want to gel purify a PCR product - what is retained? What is discarded? Gel purifying a PCR product selectively retains the target DNA fragment while discarding the unwanted bits which results in a purified sample. Week 4 1) What are some of the disadvantages of cloning with restriction enzymes and ligase? How does the Gateway approach overcome those disadvantages? Some possible disadvantages to traditional cloning with restriction sites is that some plasmids don’t have sites meant for multiple clones, the restriction sites that would be used for cloning might be in the gene of interest, and the whole process can be inefficient for certain DNA fragments or vectors. Gateway cloning overcomes these disadvantages because once an entry clone is obtained and the gene of interest is set, they can be shuttled into destination vectors for multiple different features and outcomes. This eliminates the need for restriction sites, increases efficiency, and minimal sequence alterations have to be made. 2) Illustrate the life cycle of lambda phage. a. Starts with a bacterial cell and a phage. b. The phage lands on the bacteria, and it injects its DNA. c. It can exist as a separate plasmid, or it can insert itself back into the bacterial genome and remain dormant (lysogine) d. Once it receives a signal, the phage DNA can excise itself from the bacterial DNA. e. Forms new phages and lyse the cell. f. Go and infect other bacterial cells. 3) Diagram the reactants and products for a BP reaction. What is the role of the att sites in the reaction? What is the name of the enzyme mix that catalyzes the reaction? For a BP reaction the reactents and products are shown below. During a reaction the attB sites in the PCR products reacts with the attP sites on a donor vector to create attL and attR sites. The enzyme mix that catalyzes the reaction is typically a recombinase enzyme like BP clonase II. 4) What is the molecular basis for how the ccdB gene product works as a selectable marker? The ccdB gene works as a selectable marker based on its toxicity to host cells and helps to get rid of unwanted bits of DNA after recombination. 5) What aspects of the Gateway system remain unclear to you after having completed these activities? I understand the background of what Gateway systems do and how they work, I think it will just be really interesting to actually utilize it in a lab space. WEEK 5 1. Why is RNAse added to buffer P1? What effect does the resuspension buffer (Buffer P1) have on the cells? RNAse is added to buffer P1 because it degrades the RNA present in the sample ensuring only the isolated DNA is present. Buffer P1 helps in resuspending the cells and disrupting the cell membrane and releasing cellular contents. 2. What are the two active ingredients of the lysis buffer (Buffer P2)? What effect do those ingredients have on the cells? Why is the pH of the lysis buffer important? The two active ingredients in the lysis buffer are SDS and NaOH. SDS solubilizes cellular membranes and denatures the proteins while NaOH denatures DA, helping to release it from cellular proteins. The pH of the lysis buffer is important because it affects the efficiency of DNA binding to the silica membrane of the spin column. 3. Why do you suppose the cells are incubated in lysis buffer for no more than 5 minutes? Why don't you vortex after addition of buffer P2? Cells are incubated in lysis buffer for no more then 5 minutes because after those 5 minutes there could be DNA shearing. Vortexing after the addition of buffer P2 will result in the shearing of the DNA. And could possibly reanneal. 4. What are the active ingredients of the neutralization buffer (Buffer N3)? What effect do those ingredients have on the solution? The active ingredients in Buffer N3 are acetic acid and Potassium acetate, they help return the pH to neutral allowing he DNA strand to renature. 5. After the 10-minute centrifugation, what is in the pellet? What is in the supernatent? After centrifugation the plasmid is in the supernatant and the pellet contains of the proteins lipids and DNA that precipitated out. 6. The spin column works in much the same way as the spin columns you used in week 3 for the gel purification. Do you suppose that the supernatent from the previous step contains high salt or low salt? Why? High salt, because the plasmid will bind to it in the silica membrane. 7. We won't use buffer PB. Under what circumstances might you use buffer PB? Do you suppose buffer PB contains high salt or low salt? Why? To wash the DNA bound to the spin column, it likely contains high salt. 8. What is the purpose of buffer PE? Do you suppose buffer PE contains high salt or low salt? Why? Why is it important to remove any residual buffer PE with an additional spin? Buffer PE is used to wash away remaining contaminants from the spin column. It contains ethanol and a low salt concentration to wash the DNA while maintaining its binding to the column. It is important to remove residual buffer PE to prevent ethanol to carry over. 9. Do you suppose buffer EB has high or low salt? Why? Why do you suppose the columns are incubated with buffer EB for 5 minutes before centrifugation? Buffer EB likely has a low salt concentration to elute the purified DNA from the spin column efficiently. Columns are incubated with buffer EB to ensure complete eution of DNA from the column matrix before centrifugation. 10. Summarize the steps involved in preparing plasmid DNA using the QiaPrep Sping Miniprep Kit and a microcentrifuge. NOTE: this activity is not for me! It’s to give you an opportunity to think through the protocol before entering it into your lab notebook and before coming to lab so you can avoid mistakes. a. Resuspend cells in Buffer P1 containing RNAse. b. Add Buffer P2 to lyse cells and denature proteins. c. Neutralize the lysate with Buffer N3. d. Centrifuge to pellet cell debris, leaving DNA in the supernatant. e. Transfer supernatant to a spin column and centrifuge to bind DNA. f. Wash the column with Buffer PE to remove contaminants. g. Centrifuge to remove residual wash buffer. h. Elute purified DNA with Buffer EB. i. Incubate spin column with Buffer EB and centrifuge to collect eluted DNA. j. Proceed with downstream applications using the purified plasmid DNA. Week 6 1. Illustrate/describe the difference between dNTPs and ddNTPs and describe the role each play in a dideoxy chain termination sequencing reaction, dNTPS are the regular nucleotide building blocks of DNA, they consists of a deoxyribose sugar, a phosphate group, and one of four bases: adenine, guanine, cytosine, or thymine. Role: In dideoxy chain termination sequencing, dNTPS are used with polymerase to synthesize the new strands. The polymerase incorporates the dNTPs into the growing DNA strand during replication. ddNTPs: Modified versions of dNTPs where the 3’hydroxyl group on the sugar is replaced with a hydrogen atom. Role: ddNTPs work as chain terminators in dideoxy sequencing, and cause DNA synthesis to stop 2. List the necessary components of a dideoxy sequencing reaction, - PCR products, primers, dNTPs, ddNTPs 3. Compare and contrast PCR and cycle sequencing, PCR is used to amplify a specific segment of DNA while cycling sequencing is used to determine the sequence of a DNA fragment. PCR involves a series of temperature-controlled steps (denaturation, annealing, and extension) and cycle sequencing involves using a DNA template, DNA polymerase, and a mix of dNTPS and some ddNTPs. PCR products include copies of the target DNA segment and cycle sequencing produces DNA fragments. - Two primers used in PCR cause you sequencing both strands. Sequencing one primer DIG is a side chain where you can label the primer with dig or ddntps and you can buy an antidig antibody 4. Describe the two ways in which cycle sequencing products are separated: polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (CE), PAGE à technique used to separate macromolecules, such as DNA fragments based on size and charge, the smaller fragments move quicker than the larger ones through the gel. CEà high resolution separation technique that used capillary tubes, separates DNA fragments based on size and charge but at a higher resolution. 5. Describe how the sequencing products are labeled and visualized, The sequencing products are labeled with fluorescent or radioactive tags and loaded into gels. 6. Compare and contrast dideoxy sequencing with nanopore sequencing, Dideoxy sequencing used chain termination method where the DNA synthesis is terminated at specific points using ddNTPS and Nanopore sequencing is based on the principle of passing a single-stranded DNA molecule through a nanopore embedded in a membrane. Dideoxy sequencing involves PCR amplification, cycle sequencing, and gel electrophoresis. Nanopore sequencing involves not too much preparation and can sequence DNA directly with out the need for PCR. 7. Explain the advantages of DNA quantitation using the Qbit fluorometric assay, and diagram the steps involved in quantifying DNA using the Qbit. The Qubit fluorometric assay is widely used to quantify DNA because of its high sensitivity, accuracy and specificity. The assay steps begin with sample preparation, preparation of the Qubit solution, preparing the standard curve, calibration of the Quibit instrument, loading of samples, perform the assay, measure the results and calculate the DNA concentration. Additional Questions: Why might you do a serial dilution before plating the cells? Serial dilutions make it easier to count the colonies. A what stage does the DNA enter the cell? During the heat shock Why incubate the cells on ice? What’s the difference between transformation and competency? Competence is the ability to take the DNA up in the cell Transformation is the process of putting the DNA in the cell (the action) You transform 15 pg PUC19 plasmid into some new ultracompetent cells, which have an expected transformation efficiency of >1 x 109 cfu/µg. You use a 2 mL total transformation volume, prepare a 1/100 dilution, and plate 30 µL. The next day, you count 45 colonies. Calculate the transformation efficiency. APE à Arm 1. Go to gene files cDNA sequnces and rpimer sequences 2. Copy cDNA sequence 3. Save as name 4. Select all capital letters à label as a feature as (arm coding sequence) 5. Go to primers 6. Search for lowercase letters in ape 7. It should be right at the beginning of the gene. 8. Delete the stuff before 9. Copy upper case letters and paste right before the sequence 10. Copy reverse and hit paste revers completment 11. Featuresà featurel library à gateway features à annoted features from library 12. ARM PCR product name Scarbled protocol and put in the ordwer What does Lysol buffer do what happens if you skip it Binding prooeties of silica membrane What happens during 20 minutes on ice What is the role of calcium Tm= temperature at which half DNA is annealed and the other is denatured If you set annealing temp above te tm the primers woudnnt bind and we would see a faint band If its lower then Tm it will bind really bold and you can see smear on the gel Make primers longer and shorter to raise or lower Tm, do this from 3’ end QUIBIT is more accurate because it only binds to DNA Throw BfrIs in the folder