Molecular Genetics Lab Quiz Preps
Molecular Genetics Lab Quiz Preps BIOL 3222
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This 14 page Bundle was uploaded by Madeline Abuelafiya on Saturday January 30, 2016. The Bundle belongs to BIOL 3222 at Southern Methodist University taught by Dr. Strecker and Dr. Batista in Winter 2016. Since its upload, it has received 230 views. For similar materials see Molecular Genetics Laboratory in Biology at Southern Methodist University.
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Date Created: 01/30/16
Preparation for Quiz 2 Biol 3222 – Molecular Genetics Laboratory 1. Know the definitions of “precision” and “accuracy” as well as “in vitro”. 2. What is the difference between “microchemistry” and “sterile technique” and “microbiology techniques”? 3. Know the ranges of the micropipetters you will be using and which of the four you would use for a specific volume. 4. How do you verify that the desired volume of a solution has been drawn up into the tip of a micropipetter? 5. How do you deliver the liquid in the micropipetter tip to a new tube? 6. How do you make sure that all the liquid has been dispensed from the tip? 7. What are three ways of mishandling a micropipetter that could lead to liquid entering the barrel of the device? 8. The tip on your micropipette drips liquid without your depressing the plunger. What could be the problem? 9. What is the purpose of having a Bunsen burner (or alcohol burner) producing a flame on the bench top while you are using sterile technique (other than for flaming glassware and pipettes)? 10. Do you think it is necessary to use a Bunsen burner when you are using sterile glassware, pipettes and solutions? 11. Why should an open bottle, tube or flask always be held at an angle when adding or removing liquid using sterile technique? 12. While loading the well of a gel with loading dye, you notice that the dye goes into the well, but then goes right back up into the tip of the micropipette as you are pulling the micropipette out of the well. What could be the problem with your technique and how will you correct it? 13. While loading the well of a gel with loading dye, you notice that the dye diffuses rapidly in the buffer that covers the gel, but doesn’t go into the well. What could be the problem with your technique or the loading dye?] 14. Be sure to have completed the microfuge exercise handed out the first week of the lab course. Preparation for Quiz 4 Biol 3222 – Molecular Genetics Lab 1. Define “electrophoresis.” General technique used to separate a mixture according to size. 2. What is the driving force for electrophoresis and what does it act on? The electric field is the driving force acting on charged molecules. 3. Why is double stranded DNA especially suited to size separation in electrophoresis? Two reasons. Double stranded DNA has a very uniform structure and at any given pH there is a constant negative charge for every nucleotide in the molecule. 4. What is the relationship of the movement of a double stranded DNA molecule in an electric field relative to its mass (length in base pairs)? It is inversely proportional to its mass. 5. Make sure you understand the plot of the molecular weight ladder fragments (in kb) versus their migration distance from the loading well (which is a linear axis and which is a log axis) and how this plot is used to estimate the size in kb of unknown fragments from an agarose gel. The distance has the linear axis and the weight (kb) has a log axis. Weight ladder fragments brightness – the mass Migration distance size 6. What is agarose and where does it come from? Uncharged galactose based polymer that comes from seaweed 7. Why is agarose an ideal gel matrix for electrophoresis experiments? Because of its uncharged and unionized nature. 8. What is the purpose of the TBE buffer used both in the gel and in the electrophoresis chamber? Why is this important in the electrophoretic separation of DNA fragments? TBE buffer also fills the electrophoresis chamber and covers the gel, allowing the electricity to conduct evenly through the gel. 9. Why is EDTA used often in biochemistry and molecular biology? Is used to chelate Mg2+ ions since doing so inactivates nearly all enzymes that process or degrade nucleotides. EDTA is used to inactivate nucleotide degrading enzymes by binding to and sequestering Mg 2+. 10. Why is the charge to mass ratio of DNA relatively constant during electrophoresis? Why is this important in the electrophoretic separation of DNA fragments? DNA is negatively charged due to its phosphate residues in the backbone and because there is one phosphate per base pair in the DNA sequence, the charge to mass ratio of DNA is relatively constant. This is important in electrophoresis separation because there is a constant force per base pair being applied to each molecule. This allows the DNA to be proportionally separated in size by the agarose. 11. Why is Ethidium Bromide used in the staining of DNA (two reasons)? Ethidium Bromide is a flat, planar, multiringed aromatic compound that inserts neatly between the stacked nucleotides of DNA. It also is fluorescent in UV which allows us to visualize separated DNA bands. (1. Structure 2. Fluorescent) 12. What are the four properties that affect the speed of migration of DNA fragments through agarose gel matrices? 1) The Mass (Length) of the DNA 2) The concentration of agarose in the gel 3) The conformation of DNA 4) The applied current (milliamperes) moving through the gel 13. What are the three conformations that doublestranded DNA can adopt and how do they migrate differently in a gel. Which form migrates true to its size? Why do the other two forms of double stranded DNA not migrate true to their size? Form 1: Closed, Circular DNA supercoiling migrates faster Form 2: Nicked, Circular DNA creates a large, open circle conformation by relaxation of supercoils slower to migrate Form 3: Linear DNA true to size. 14. If you wanted to resolve DNA fragments that were larger, how would you change the concentration of the agarose in your gel and why? To resolve larger DNA fragments the gel would be a smaller percent concentration of agarose (0.60.9) to have less agarose strands ands more open solvent space for the larger DNA molecules to pass through. 15. What is bacteriophage and in what two phases can it exist in the bacterial cell? What is a cos site and what does it allow the to do? The bacteriophage is a virus that infects E.coli. It is a lysogenic temperate bacteriophage. The two phases it exists in are the: lytic phase and the quasistable prophage stage Lytic stage: causes lysis of the host cell Prophage stage: has its genome incorporated into the host chromosome so when the host replicates, so does the prophage genome. The cos site is a single stranded 12 base complimentary overhang at each end The cos site is important in phage particle packing and allows it to convert between linear and circular formation DNA. 16. What is the natural purpose of restriction endonucleases in bacteria? How then are molecular biologists able to introduce foreign DNA into strains of E. coli? The natural purpose of restriction endonucleases in bacteria is a defense mechanism to prevent the expression and take over in a cell by viral DNA. Biologists are able to introduce foreign DNA into E. coli strains because they choose E Coli which is deficient or mutated in their naturally occurring restriction endonuclease systems but are normal in their restriction methylation systems. Therefore, the E. coli will not restrict the foreign DNA but remethyl it as E. coli DNA 17. Why are Type II restriction enzymes used in recombinant DNA experiments? Type II restriction enzymes are used in recombinant DNA experiments because they are simple, stable, and have identical recognition sites. They recognize specific 4,5, or 6 base pair sequences and they are able to cleave DNA within the recognition site. Preparation for Quiz 9 (on PCR lab) Biol 3222 – Molecular Genetics Lab 1. What is the normal function of MDR genes in the cell? MDR gene transcribes multidrug resistance proteins that are MDR pumps. These give the cells resistances to many drugs and cancer therapies MDR1 generally removes toxic chemicals and compounds from normal cells in the small intestines, liver, kidney, breast, and testes since these tissues have a primary role in absorption, metabolism, and excretion. It also is important in the blood brain barrier. 2. How could a mutation in the human MDR1 gene affect the cancer treatment plan for a patient who is homozygous for a mutation in this gene? Increases drug resistance. Generally have to do with the overexpression of the pump in a patient that has been treated by a chemotherapeutic. 3. What are the basic elements contained in a PCR reaction? Taq polymerase, rna primer, DNTPs, dna template 4. What are four important properties of PCR primers? 1. They must be complementary to the boundaries of the target DNA sequence. 2. The 3’ end of the primers must be oriented towards eachother 3. The primers usually have a melting temperature of 5060 degrees C. The melting temperature of double stranded DNA is that temperature at which 505 of the SNA denatures into single strands 4. The primers must be specific for the flanking regions of the target DNA sequence and not bind to to other nonspecific sequences of DNA. Lack of specificity for the primers will result in amplification of more than one DNA sequence. 5. What is one equation used to calculate annealing T? Make sure you can apply this equation to a given primer sequence (see questions 13). Ta = Tm – 5degreesC …… T =[4mG+C) + 2(A+T)] degrees C 6. What is the purpose of the thermocycler? The thermocycler is to regulate the temperature quickly and accurately heat and cool the tubes 7. What are the three elements of a single cycle of PCR? 95 melts the dna 55 primers anneal 72 elongates 8. What contribution did Kary Mullis make to the development of PCR? Developed the polymerase chain reaction specifically “thermal cycling” and the use of thermostable DNA polymerases 9. Why is Taq Polymerase used in the PCR reaction? Taq polymerase is a DNA polymerase isolated form T.aquaticus that has an enzymatic optimum temperature of 7080 degrees C and can withstand temperatures above 90C for long periods of time. It doesn’t senature easily 10. What is one drawback of using the original Taq polymerase in PCR? One drawback to the use of the original Taq polymerase for PCCR is that is has a relatively high error rate estimated at 1 mismatch per 9000 because Taw lacks a 53’ exonuclease 11. In a PCR reaction, why is only the DNA between the primers amplified in the majority of cases after 30 cycles? Because DNA pol can only add nucleotides to the 3’ end so only the 5’ end of the primers definies the limit of the amplicon. 12. What are three advantages of using ReadytoGo PCR beads and what do they contain? Provides optimal amount of reagents needed for PCR and free of contaminating DNA. Contain Taq DNA polymerase, dATP, dTTP, dCTP, and dGTP and a buffer and salt for a typical 25ul PCR reaction. 13. If you were given a sequence of a PCR primer, what simple equation would you use nd to calculate the annealing temperature that should be used in the 2 step of each cycle of the PCR program? Ta = Tm – 5degreesC …… T =[4(m+C) + 2(A+T)] degrees C Tm= when have of the DNA is single stranded and half is double stranded 5. How do the two plasmids differ from each other: pWS4.5 and pBluescriptIISK? (DNA sequence, protein expression and colony differences). 1bp, functional lac Z B galactosidase expression, blue vs white colonies 6. How are Primers 1 and 2 used to make the nucleotide change to restore Bgal activity to pWS4.5? 8. Why are the new copies from the pWS4.5 template NOT used as template in subsequent reactions? They are not methylated this allows for the correction of mismatch repair 10. Describe the mutation you will be reverting back to wildtype in this experiment. How will you recognize this event? Reverting genetic loss of function of the lac Z gene back to wildtype. We mutanted a nonsense mutation within the coding sequence of the Bgalactosidase gene, lac Z in the plasmid. This created truncated proteins not allowing functional B galactosidase. The mutation within the lac Z gene specifically was a CT transition mutation that converted a functional codon for a glutamine residue into a nonsense stop codon. (CAA TAA). We can recognize this event because if there is no functional lac Z, the colonies will be white when grown on LBAX. If lac Z is functional, the colonies will appear blue 11. You are plating four different DNA samples on LBAmp plates: QC = 10 μl of the QuikChange reaction (no DpnI treatment) Blue and white colonies – if white colonies are seen, then the quick change reaction failed and you did not get any of your plasmid QC+ = 10 μl of the QuikChange reaction with DpnI treatment Only blue colonies – control there is active Bgalactosidase and functional LacZ on LBAX pWS4.5 = 10 μl pWS4.5 plasmid only white colonies – confirms the nonsense mutation occurred creating a truncated Bgalactosidase from the nonfunctional lacZ gene. The transformation to a stop codon was successful. pBLU = 10 μl pBLU plasmid only blue colonies – control there is active Bgalactosidase and functional LacZ on LBAX What results (in terms of colonies – blue versus white) do you expect on each plate? What information does each plate give you about your experiment? 12. In the transformation of DH5alpha cells, what plasmid was used to make the positive control, negative control and experimental transformation? 13. If you observed blue colonies on the positive control Xgal plate, but not on the negative control and experimental Xgal plates, what would be your conclusion about the transformation and in vitro mutagenesis? The transformation and invitro mutagenesis was unsuccessful and lac Z remained functional so no truncated proteins occurred and the desired plasmid was not created Preparation for Quiz 6 Biol 3222 – Molecular Genetics Laboratory 1. What does the term “miniprep” mean in the context of this laboratory? A preparation system meaning a plasmid DNA preparation that is expected to yield ug quantities of a plasmid from a couple of ml of cell cultures. It is a robust technique. 2. Compare the QIAspin Miniprep procedure to the method of preparing plasmid from a bacterial culture in the past. (In other words, what step does it share with the old method and what new improvement has the QIAspin made on the old method). In both methods we did an alkaline lysis of the bacterial culture to release the plasmid DNA but the the method has a different way of purification which involves binding to activated silica and undergo many rounds of centrifugation. 3. What observation is the QIAspin Miniprep procedure based on? 4. In the procedure you used in the QIAspin miniprep, a. How were the cells lysed? The cells were lysed with detergent and the sodium hydroxide base, a pH neutralizing K+ acetate solutuion. b. What were the three effects of adding Potassium acetate solution to the cellular lysate? 1)neutralizes the pH 2) helps get rid of the detergent used to solubilize the membrane of the cells. 3) the potassium acetate raisws the salt salt concentration of the cell lysate to a very high volyume. c. Why is it important NOT to vortex the neutralized lysate prior to microfuging it? Vortexing would shear the long chromosomal DNA into short, plasma sized fragments d. After this microfuge spin, what is contained in the supernatent and what is contained in the precipitate? Supernatent: plasmid DNA Precipitate: cellular debris and chromosomal DNA e. When the supernatent is poured over the QIA spin column, what binds to the silica matrix? Plasmid DNA binds to silica matrix and is separated from the cellular contents that pass through the column f. How is the plasmid DNA released from the column? The pure plasmid DNA is eluted with a small volume of low ionic strength buffer or distilled water. The low salt conditions release the DNA from the silica and a final spin into a fresh, clean, microfuge tube removes the plasmid DNA from the column and deposits it pure and clean into a new tube. 5. Know the names and functions of the buffers you used with the QIAprep column and the order in which they are used. 6. In what form is your plasmid DNA when it is eluted from the column (linear or covalently closed, relaxed or supercoiled?) What protects the plasmid DNA from being damaged (nicked) during purification? When the DNA is eluted it is in covalently closed supercoiled form. The DNA is bound to the silica matrix while being purified, there is very little damage to the DNA. Preparation for Quiz 7 1. Be familiar with the definition of molecular cloning. Molecular cloning of DNA: the process of making many, many copies of a specific DNA sequence. 2. What are the five steps in a molecular cloning experiment? 1) DNA isolation (of the ion plasmid that contains the tetR gene) and preparation for cloning (preparing the ends of the DNA to ligate them into the DNA vector using restriction endonucleases) 2) The vector is prepared by cutting with HINDIII and ECORI allows the vector to be inserted in a specific orientation. (Force Cloning) 3) Covalently attach the desired DNA fragment into the restricted DNA vector with an enzymatic reaction of DNA ligase from the T4 bacteriophage (ATP dependent) 4) High efficiency transformation allows the efficient uptake of DNA into the bacterial cell. (high transformation efficiency) 5) The “correct” clone that has the desired DNA sequence in its vector must be identified from all the other clones that contain the undesired fragments or re ligated original vector. a. The first identification: on the bacterial plate recognizing the fragmented lacZ gene by inactivating the Bgalactosidase (Xgal). Correct clones will be white/colorless b. The second identification: direct selection by plating some of the transformed cells on ampicillin and tetracycline containing plates. Only cells with both antibiotic resistance genes will grow on the plate c. The third identification: isolate the plasmid DNA from some of the white colonies. After looking at the agarose gels for size screening and recognizing banding patterns of the ECORI and HINDIII digestion, we will be able to positively identify the desired clone. 3. What plasmid is the source of the DNA that will be used as the insert DNA in your cloning experiment? pACYC184 plasmid that contains the tetracycline resistance gene. 4. Which enzymes will be used to generate the desired insert fragment? HINDIII and ECORI 5. What selection method do you think will be used to recognize the presence of this fragment? The second identification method: plating the transformed cells on ampicillin and tetracycline containing plates. Only cells with both antibiotic resistance genes will grow on the plate. 6. Which plasmid will be used as the cloning vector for this experiment? pUC119 7. How will you prepare this plasmid to receive the insert DNA? The ECORI and HINDIII sites in the multiple cloning sites of the pUC119 vector will be used to force clone the fragments into one specific orientation in the lacZ’ gene of the vector. (using restriction endonucleases) 8. How will you covalently link the insert DNA to the cloning vector? DNA ligase (ATP dependent) Preparation for Quiz 5 – Biol 3222 Molecular Genetics Lab 1. What definition did Frederick Griffith give to the term: transformation, and how did he observe this phenomena while working with Rstrains and Sstrains of bacteria? Transformation is how bacteria are capable of transferring genetic information. When living Rstrains of bacteria were exposed to heat killed Sstrains before being injected to the mouse, the Rstrains could be transformed into pathogenic Sstrains and that the mouse would die of pneumonia. 2. What does “competence” mean and what is the difference between “artificial” and “natural” competence? Competence means state in which species are able to do what they are supposed to do. Natural competence is when species are naturally able to take up DNA from their environment. Artificial competence is when species are treated with reagents and procedures to dramatically increase their competence. 3. (See ppt lecture from last week) What are the four basic steps in a transformation experiment and what is happening in each step? Preincubation: Bacterial cells are suspended in a solution of cations and incubated at 0 C. Molecular diffusion occurs. Incubation: DNA is added. The low temperature congeals the cell membrane, stabilizing the distribution of charged phosphates across the cell membrane. Heat Shock: The cell/DNA suspension is briefly incubated at 42 C and then returned to C. This causes one or the bacterial cell takes up more DNA molecules bound to the surface of the cell. o Recovery: LB broth is added to the DNA/cell suspension and incubated at 37 C before plating on selective media. Transformed cells recover from the treatment, amplify the transformed plasmid, and begin to express the antibioticresistance protein. 4. What are the four specific sequences a cloning vector should contain and what is the function of each? Replicons: direct replication of DNA Selectable gene or gene product: necessary to distinguish between the appx 11000 cells that take up the cloning vector in the transformation. Unique cleavage site for restriction enzymes: makes the reproducible insertion of foreign DNA into specific sites in the plasmid possible. Suitable expression control elements: allow normal levels of gene expression 5. What is a Multiple Cloning Site and where is it usually positioned in a cloning vector? What advantage does a MCS sequence give an investigator who wants to clone a particular gene into a cloning vector? Multiple Cloning Sites are regions of unique restriction site sequences that have been genetically engineered into the vector in a very short region of DNA. They are usually places near the very beginning to the protein coding sequence. This region is non essential so short insertions don’t change the reading frame. It gives the advantage of added restriction sites without functionally inactivating the gene product. 6. In the pUC119 vector, what does the LacP gene allow the investigator to do with an inserted gene? What is the purpose of the MCS sequences being located within the LacZ’ gene? LacP(transcriptional promoter) in pUC119 allows the LacZ to be promoted (because LacP is upstream). Cloning foreign DNA into the MCS of this plasmid will allow transcription of the inserted DNA. Therefore the desired strand of dna will be used as a template and promoted by the Lac P. 7. In the pACYC184 vector, what are the two means of selection for successful transformation? Why is a low copy number plasmid advantageous in situations when the investigator is expressing protein from the cloning vector? The two means of selection for successful transformation are chloramphenicol and tetracycline. Low copy number is advantageous because it doesn’t result in too much membrane protein for the membrane to accommodate and does not cause aggregations 8. (See ppt. lecture from last week) Be familiar with the three different ways bacteria can acquire DNA from the “environment” (or other bacterial cells). Bacterial plasmids, antibiotic resistance, LacZ gene Transduction, conjugation, and transformation Transformation uptake dna via environment Transduction virus Conjusugation in between bacterial vells
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