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Homework 3 Based on parts of labs 9 -14 18% of final grade Please use the same headings as used below. Submit to Turnitin via Ted. Include the...

Design 20 nucleotide long primers to amplify luxC of accession number AF170104 so that the primers are within 200 basepairs of the start and end of the luxC gene. The PCR product should contain the RBS and start and stop codons for luxC. You can do this by eye or you can use an online program like Primer 3. Make sure the primers you design have approximately the same number of G’s and Cs, so that their Tms are at least 50 oC and are within 5oC of each other. (They do not have to end in a C or C.)
Homework 3 Based on parts of labs 9 -14 18% of final grade Please use the same headings as used below. Submit to Turnitin via Ted. Include the carbons from labs 10 -14 . Part 1. Cloning lux AB into DH5alpha cells Introduction (1page double-spaced): Explain the strategy you used to clone lux AB into E. coli, starting from the PCR reaction you did in lab 8 through to screening colonies . Also explain the function of the proteins encoded by the lux AB genes, and how you planned to identify positive colonies. If you like, you can make a flow chart to outline what you did. Combined Methods, Results and Discussion 1. Generating the PCR product containing l ux AB (lab 9 and 10): a. Briefly explain how you generated the PCR product containing lux AB – that is, what did you use as the template for your PCR reactions. (Do not explain the theory of PCR!) b. W here did the primers anneal relative to the lux AB genes? What is the expected size of the PCR product? (You will need to go back to your bioinformatics exercise from lab 9 to answer this.) c. You do not need to show the recipe for the mastermix, but include the settings we used on the thermal cycler we used to do the PCR (denaturation setting, annealing, etc.). d. You do not need to include the picture of your gel from lab 10, but comment on whether or not you got a PCR product of the expected size in your reactions and what the yield of your PCR product was. 2. Digests (lab 11) a. Explain what samples M, N, and O contained. b. Explain why you digested the PCR product and pGEM with Xba l and Eco Rl, instead of just one enzyme like we did for the genomic library. 3. Clean up of pGEM to remove stuffer fragment and gel of digests (lab 12): a. Explain why you removed the stuffer fragment from half of the digested pGEM prep. b. Include the gel picture from lab 12 labeled as usual, and explain the results from the gel in the text. c. Make a table showing the amounts you estimated were in the DNA bands in the lanes containing samples M, O and P and the calculated concentrations in samples M, O and P. 4. Ligations (lab 12) a. List the ligations you did (the chart on page 112 should be in your carbons). b. Draw a diagram of the recombinant plasmid you will create by ligating the PCR product into pGEM. Label where the Xba1 site and EcoR1 sites in the recombinant vector are relative to the lac Z alpha promoter and the lux AB genes. Also, show
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where the RBS, and start and stop codons for lac Zalpha, lux A and lux B are relative to one another. 5. Transformation results (lab 13-14) Present and summarize the results of your transformations in a table, with columns for the numbers of blue and white colonies, and the percent white colonies similar to that on page 80 of the manual. Then discuss the success of your experiment in general, and include answers to the following questions: a. What was the transformation efficiency of your competent cells? b. Did removing the stuffer fragment make any difference in terms of the number of white versus blue colonies you got? Why might you expect it to? c. Did varying the insert to vector ratio make a difference in terms of how many blue versus whites you got? Why might you expect it to? d. What might be an advantage of cloning your PCR product using the 3’A overhangs created by Taq and a T-tailed vector instead of cloning the way you did? What might be a disadvantage? Part 2. Design PCR primers to amplify lux C. 1. You want to do some studies on the structure of the lux C protein, and so you want to clone the gene and produce the lux C protein in E.coli . a. Design 20 nucleotide long primers to amplify lux C so that the primers are within 200 basepairs of the start and end of the lux C gene. The PCR product should contain the RBS and start and stop codons for lux C. You can do this by eye or you can use an online program like Primer 3. Make sure the primers you design have approximately the same number of G’s and Cs, so that their Tms are at least 50 o C and are within 5 o C of each other. (They do not have to end in a C or C.) b.Use the Wallace formula or this website http://www6.appliedbiosystems.com/support/techtools/calc/ to calculate the Tms and show the primer sequences and the Tms in a table. Make sure you show the primers 5’ to 3’. 2. Blast your primers and copy and paste the alignments of where the primers anneal in the lux operon entry accession number AF170104.1 into your homework. Include whether the primers are +/+ or +/- to the strand shown in NCBI. How big is your PCR product? 3. a. Now you want to clone your PCR product into pGEM by adding restriction enzyme sites to the 5’ end of the primers and then digesting the PCR product and pGEM with those enzymes. One of your labmates, Bill, suggests you use add Hincll site to the 5’ end of the forward primer and Pst 1 site in the reverse primer. Another labmate Joe suggests you use Sal1 in the forward primer and Pst1 in the reverse primer. (Note – you have to make sure those restriction sites are not in the lux C sequence. To do these, put the lux C sequence into NEB cutter and find the enzymes that cut within the sequence. Look back at the bioinformatics exercise in lab 9) b. Now go look at the pGEM map at the end of the manual. You want to use the promoter for Lac Zalpha to drive transcription of your inserted lux C gene. Do you think you should use the
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