BC2004 Review Sheet for Lab Exercises 7-11 Spring Semester 2005

BC2004 Review Sheet for Lab Exercises 7-11 Spring Semester 2005 Lab Exercise 7 Drosophila crosses, three weeks Vocabulary: phenotype, genotype, gene, allele, locus (loci), sex chromosomes, autosomes, homozygous, hemizygous, and heterozygous. Describe Mendels s law of segregation and his data that supports it. Describe Mendel s law of independent assortment and his data that supports it. How can you tell the difference between male and female Drosophila? How can you remove Drosophila from the vials without letting them all fly away? Explain precisely how you set up your two crosses one for C, P, D and the other for your mutant trait Explain why you removed and discarded the parental flies after one week together in the vial Did you analyze F1 or F2 data from your crosses? How could you have set up your experiment so that you could have collected F2 data? How much earlier in the semester would you have needed to begin the experiment? How are the virgin female flies collected? Where do they come from and why does it occasionally happen that there might be a non-virgin female in the vial labeled virgin females? How did you make your predictions of F1 offspring ratios for your crosses? You should be able to set up crosses on paper and make predictions given different genes and different sets of circumstances. How can you tell which alleles of multiple genes were originally on the same chromosomes in the parent flies? How can you determine whether genes are linked or not? (And, physically what does linkage mean?) Why is it unlikely that your data reflected a perfect match to your hypotheses for the fly data? What is the most likely explanation for the large number of wild type F1 offspring for your wt x CPD crosses? How can you tell whether a gene is on a sex chromosome or an autosome? How could you tell whether a gene on a sex chromosome is on the X-chromosome or the Y- chromosome? What is meant by the phrase reciprocal crosses? Explain under what circumstances you should use a Chi-square statistical analysis. What is the mathematical formula for computing a Chi-square value? Be able to calculate expected phenotypic frequencies and Chi-square values (you ll be given a probability table to use in the exam). How can you determine p-values (and what is a p-value?) after you have calculated a Chi-square value and degrees of freedom? What would it mean if your Chi-square value were zero? Assume that you determined a p-value of 0.2 for your Chi-square test of a 3:1 hypothesis. Describe precisely what this p-value would mean with respect to your hypothesis. ** What are the parts of a formal lab report? Briefly, what belongs in each section? BC2004, Spring Semester 2005, Final exam review sheet and practice problems-1

Lab Exercise 8 Bacterial transformation, two weeks Vocabulary: transformation, competent, vector, plasmid, recombinant, transformant, lawn, and transgenic. Define transformation (note that it is not specific to bacteria; it is possible to transform other kinds of organisms too). Describe the phenomenon of lateral or horizontal gene transfer. How does it differ from vertical gene transfer? What does it mean for an organism to be competent for transformation? The E.coli cells that you transformed in lab are not naturally competent. How did you make them competent? What is the difference between a vector and a plasmid? Are there vectors that are not plasmids or plasmids that are not vectors? Describe the roles of the proteins encoded by the amp R and lacz genes. How did you select transformants after transformation? Did all or most of the cells you tried to transform successfully become transformants? Describe X-gal and name the enzyme that converts it to a colored product. What color is the product? Be able to make predictions of relative colony numbers and colors of E.coli populations grown on different media. How can you make serial dilutions? Why make serial dilutions? What is the dilution factor for a 10-3 plate? For a 10-5 plate? By counting colonies on plates, you recorded 85 colonies on your 10-4 plate, 810 colonies on your 10-3 plate, and 9 colonies on your 10-5 plate. Which of these data points should you use for further calculations and why? How can you calculate the number of E.coli cells that you attempted to transform? How can you calculate the percentage of cells that you attempted to transform that were successfully transformed? How can you tell which colonies received pblu plasmid and which received recombinant pblu plasmid (in which the lacz gene has been interrupted by a PCR product amplified from lambda? What is a colony? From what does it form? What does cfu mean? Physically, what is a cfu? Why did each group of students have different numbers of colonies on its plates? How could the lab exercise be changed so that there would be more uniformity in the number of colonies among groups? Given a restriction map of pblu, and the length of the PCR product inserted into the lacz gene, you should be able to predict the sizes of restriction digests of the pblu plasmid. (map is at the bottom of the web page at http://www.dnalc.org/resources/nucleotideseqs.html. You should also be able to do this for the other plasmids mapped on that page (or any other plasmid too). BC2004, Spring Semester 2005, Final exam review sheet and practice problems-2

1. Predict the outcomes of transformations of E.coli with the following plasmids: tet R pred lacred tet R pred recombinant form A lacred PCR product inserted here, interrupting lacred gene PCR product inserted here, interrupting lac tet R gene tet R PCR product inserted here, interrupting tet R gene tet R PCR product inserted here, interrupting lacred gene pred recombinant form B lacred pred recombinant form C lacred You will use separate samples of these purified plasmids to transform E.coli. How would you do this? The tet R gene confers resistance to tetracycline (an antibiotic) (the E.coli are naturally suseptible to tetracycline). The (imaginary) lacred gene encodes a protein that catalyzes the conversion of the substrate R-gal to a red product. Predict the outcomes of transformation with each plasmid on the following media: PCR product inserted here, interrupting some noncoding DNA sequence tet R pred recombinant form D lacred LB media: Non-transformed? Transformed with the non-recombinant pred plasmid? Transformed with recombinant form A? Transformed with recombinant form B? Transformed with recombinant form C? Transformed with recombinant form D? LB + R-gal media: same as above LB + tet media: same as above LB + R-gal + tet media: same as above A table is provided for your predictions on the next page. Predict relative number of colonies and their color(s). BC2004, Spring Semester 2005, Final exam review sheet and practice problems-3

LB media LB + R-gal LB + tet LB+R-gal+tet nontransformed E.coli E.coli transformed with nonrecombinant pred plasmid transformed with recombinant form A plasmid transformed with recombinant form B plasmid transformed with recombinant form C plasmid transformed with recombinant form D plasmid BC2004, Spring Semester 2005, Final exam review sheet and practice problems-4

Lab Exercise 9 PCR, two weeks Vocabulary: DNA structure, 5 -end, 3 -end, phosphodiester bonds, and antiparallel. Draw the structure of a dinucleotide made up from the nucleotides datp and dctp (you don t need to be able to draw the bases themselves, just use A and C.). How would the structure different if the dinucleotide was made from of ATP and CTP? Draw anti-parallel strands of DNA. What holds the two strands in a double helix together? What is meant by the term semi-conservative replication? To which end(s) of a strand of DNA can DNA polymerase add nucleoside triphosphates? From precisely where does DNA polymerase get the energy it needs to crease a new phosphoester bond? Describe why primers are necessary for the process of PCR and why external, custom-made primers (at $1 per base) are not required in vivo (i.e. in living systems). Physically, how do PCR primers and in vivo primers differ? Where do in vivo primers come from (which enzyme(s) polymerizes their construction and what is used a the template?)? Why did you use carefully designed, custom-made primers for your PCR reactions? Why not use just any old primers that you might find in a molecular biology freezer somewhere? (How do pairs of primers differ from each other?) Why do you need a primer pair made up of a forward and a reverse primer for PCR? Why not use just one primer? Describe the role of the template DNA in a PCR reaction. Precisely what is using the template DNA as a template and what is it a template for? The beginning of the DNA sequence that is complementary to the forward primer and the end of the DNA sequence that is complementary to the reverse primer that you used in lab are 1,028 base pairs apart. Predict the length of the PCR product that can be amplified using this primer pair. Precisely why, when this PCR product is inserted into a restriction site in the lacz gene, does the lacz gene no longer encode a functional β-galactosidase enzyme? Think about the processes of transcription and translation. How can you use PCR to amplify 6 different genes? Would you need to set up 6 different PCR reactions or could they all be done in the same tube? Why? What ingredients would be necessary for the amplification of each of those genes and what is the role of each of the ingredients in the reaction(s)? How would the ingredients differ if you wanted to amplify gene 1 in tube 1 and gene 2 in tube 2? What are the three temperatures and times of the main steps in a PCR run? What happens to the DNA during each of these steps? Why repeat these three steps in sequence many times? Which of these two temperatures are generally the same regardless of the product being amplified? Why does the third temperature need to be specific for each specific product being amplified? (You are strongly encouraged to review the Worksheet for PCR that was distributed during recitation those questions are not repeated here, but they are good ones!) Could you use poodle DNA polymerase to carry out PCR? What about goldfish DNA polymerase? Why or why not? Even though you wanted to amplify just one PCR product in lab, why did you set up two PCR reaction tubes? How did your reaction tubes differ from each other and what did you predict for the outcome of each reaction? BC2004, Spring Semester 2005, Final exam review sheet and practice problems-5

2. Design primers to amplify this region of chromosome 6 from platypus (I m making this up ). You ll want a forward primer and a reverse primer. Your budget is $34 (each primer base costs $1 from the custom primer company). 5 - A T T G C C G A A T G C C A A T G C A T 3 3 - T A A C G G C T T A C G G T T A C G T A 5 Your forward primer: Your reverse primer: 3. You decide that you love the pred plasmid and can t bear to not keep working with it. You design primers to the non-recombinant plasmid sequence that amplify the tet R gene and the lacred gene. The size of the PCR products are 1000 bp for the tet R gene and 3000 bp for the lacred gene. The inserts from the bacterial transformation problem are 500 bp each. Predict the results of PCR reactions using the two pairs of primers (in separate PCR reaction tubes, why?) non-recombinant pred : Amplification of tet R? If so, what size (in bp)? Amplification of lacred? If so, what size (in bp)? recombinant form A: Amplification of tet R? If so, what size (in bp)? Amplification of lacred? If so, what size (in bp)? recombinant form B: Amplification of tet R? If so, what size (in bp)? Amplification of lacred? If so, what size (in bp)? recombinant form C: Amplification of tet R? If so, what size (in bp)? Amplification of lacred? If so, what size (in bp)? recombinant form D: Amplification of tet R? If so, what size (in bp)? Amplification of lacred? If so, what size (in bp)? BC2004, Spring Semester 2005, Final exam review sheet and practice problems-6

Lab Exercise 10 Restriction Digests, two weeks What is a restriction endonuclease enzyme? Why are restriction enzymes so named? What do they restrict? What is the difference between an endonuclease and an exonuclease? (This question might be difficult to find the answer to: exonucleases cleave nucleotides off of the end of polynucleotides; endonucleases cleave somewhere inside a strand of polynucleotides.) Restriction enzymes are part of a restriction-modification system of bacterial defense; what is the modification part of the system and why is it necessary? What is the difference between a bacteriophage and a bacterium? Describe the 5 steps in a generalized bacteriophage life cycle. Which step(s) does restriction digestion prevent? What step does an open cold sore represent and what is it spreading? Can bacteriophage live on their own? If not, whose machinery do they use to accomplish DNA replication, transcription, and translation? Describe what is meant by the term restriction recognition site. Do all restriction enzymes have the same restriction recognition site? Precisely what bond(s) do restriction enzymes cleave? How do restriction enzymes get their fancy names (e.g. EcoO1091)? What is the difference between sticky and blunt ends? Which are generally more useful in the molecular biology laboratory and why? What does the enzyme ligase do and why is it necessary in the production of recombinant DNA? What were the steps required to get that PCR product inserted into the pblu plasmid? What kind(s) of enzymes were needed to accomplish this? Of what does a restriction map show the relative location? How can you calculate a restriction map? What ingredients should you add to a restriction digestion reaction and what role does each ingredient play? The elongation temperature in PCR is always 72-degrees C; the incubation temperatures of restriction digests vary. Why? The following is a restriction map of the BamH I, EcoR I and Hind III sites in the lambda genome. Why aren t the pieces the same sizes you saw in the restriction map of the Apa I and EcoO 1091 sties? Given this map, predict the sizes of the restriction digest fragments (the pieces after cutting) for digests with just Bam HI, just EcoR I, just Hind III, double digests with pair wise combinations of two enzymes (B+E, E+H, and H+B), and a triple digest (B+H+E). Actually, when I looked up the real map for these sites, it turns out that it is too complicated to be answered easliy. It would take me an hour to make the map on this handout and a long time for you to do the calculations. Instead, I ve made up some easier example problems below. You should understand the concepts of how to do these kinds of calculations. Practice the problems given on the following pages and visit the website: http://www.carolina.com/biotech/plasmid_problems/plasmid_guide2.asp for more practice drawing restriction maps given data rather than predicting data when given the map. There is also an introduction and a sample problem with a step-by-step solution guide. BC2004, Spring Semester 2005, Final exam review sheet and practice problems-7

4. Digest of LINEAR piece of DNA whose map is shown below: 1000 bp B 500 bp H 2000 bp H Give the estimated sizes of the restriction fragments from the digestion reactions below: Enzyme B alone: Enzyme H alone: Enzymes B and H together: Draw a gel (including some DNA markers) that shows the approximate results you would see for these digests. Note that you would only see 1 band in the H only digest; why? 5. Digest of LINEAR piece of DNA whose map is shown below: 1000 bp E 200bp P 2000 bp E 800 bp Give the estimated sizes of the restriction fragments from the digestion reactions below: Enzyme E alone: Enzyme P alone: Enzymes E and P together: Draw a gel (including some DNA markers) that shows the approximate results you would see for these digests. BC2004, Spring Semester 2005, Final exam review sheet and practice problems-8

6. Try a map of a circular plasmid: A 300 bp B 300 bp A 1200 bp Give the estimated sizes of the restriction fragments from the digestion reactions below: Enzyme A alone: Enzyme B alone: Enzymes A and B together: Draw a gel (including some DNA markers) that shows the approximate results you would see for these digests. BC2004, Spring Semester 2005, Final exam review sheet and practice problems-9

7. Try a map of a different circular plasmid: A 200 bp C 800 bp 4000 bp B Give the estimated sizes of the restriction fragments from the digestion reactions below: Enzyme A alone: Enzyme B alone: Enzyme C alone: Enzymes A and B together: Enzymes B and C together: Enzymes C and B together: Enzymes A, B, and C together: Draw a gel (including some DNA markers) that shows the approximate results you would see for these digests. BC2004, Spring Semester 2005, Final exam review sheet and practice problems-10

8. This time, start with the data and draw the map. LINEAR piece of DNA (why do you need to know this information?) What is the total length of this piece of DNA and how do you calculate this? How many H sites are there? How many B sites are there? H: 1200 bp and 2800 bp B: 800 bp and 3200 bp H+B: 800 bp, 2000 bp, and 1200 bp 9. Again, start with the data and draw the map. CIRCULAR piece of DNA (why do you need to know this information?) Total length? How many B sites are there? How many H sites? How many P sites? B: 200 bp, 5000 bp H: 5200 bp P: 5200 bp B+H: 200, 1400, 3600 bp P+H: 600, 4400 bp B+P: 200, 3000, 2000 bp B+P+H: 200, 600, 1400, 3000 bp BC2004, Spring Semester 2005, Final exam review sheet and practice problems-11

Lab Exercise 11 Agarose gel electrophoresis. Define the following terms: agarose, gel, electrophoresis, and polymerization. Do larger/longer or smaller/shorter pieces of nucleic acid move faster through the gel? Why? What is meant by the phrase molecular sieve? Are nucleic acids (in a neutral or basic environment) negatively or positively charged? Precisely where are they charged? Are nucleic acids attracted to the positive or negative electrode during electrophoresis? If you wanted to separate pieces of DNA that were 1000 bp and 2000 bp, should you use a higher or lower percentage of agarose in your gel than if you wanted to separate pieces that were 100 bp and 200 bp? Why? How much buffer should you pour into your gel rig and why? What forms the wells in your gel? What are the ingredients in the loading buffer that you add to your DNA samples before loading them in the gel? What is the role of each ingredient? Why did you need to heat your restriction digest fragments for 10 minutes at 65-degrees C before loading them on your gel? Why should you not have taken the samples out of the water bath and let them sit at room temperature for 20 minutes before loading the gel? Why didn t you need to heat the PCR samples or the DNA markers? When loading a gel, you shouldn t push the micropippetor plunger down to the second stop; why not? Why couldn t you run your gel at 236 Volts to have it go twice as fast so that you could finish lab in half the time? Why can t you see the bands of DNA under the fluorescent lights in the lab rooms? What did you have to do to visualize where the DNA bands were located and why? Physically what is a band on a gel? What does it represent? What did you measure in order to get the data to plot on your standard curve? What information did you need to have given to you? Why do you plot the standard curve from DNA markers from agarose gel electrophoresis on semilogarithmic graph paper? What is semilogarithmic graph paper? Why didn t you plot your standard curves using Biuret reagent (protein concentrations, remember?) on semilog graph paper? In your agarose gel electrophoresis standard curve, what were the units on the x-axis? the y- axis? Why should you ALWAYS and under ALL circumstances label all axes? Why didn t you find any 29-bp bands on your gels? Please refer to all the questions at the end of the lab exercise 11 handout, on pages 11-14 through 11-18. Not all of these questions are repeated here, but you should check that you understand their answers because your lab notebooks will not be corrected before your final exam. (Remember that your lab notebooks are due May 3 rd.) BC2004, Spring Semester 2005, Final exam review sheet and practice problems-12