Your mutagenized sample showed 523 CFU after plating 100uL of media on the 10^3 dilution plate. The control, unmutagenized, sample showed 417 CFU after plating 100uL of media on the 10^8 dilution plate. Calculate the % survivorship for this mutagenesis experiment.
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Your mutagenized sample showed 523 CFU after plating 100uL of media on the 10^3 dilution plate. The control, unmutagenized, sample showed 417 CFU after plating 100uL of media on the 10^8 dilution plate. Calculate the % survivorship for this mutagenesis experiment.
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- Cloning vector, vj, is 90 kbps (kilobase pairs) long, and contains two cleavage sites recognized by ECORI (restriction endonuclease) as indicated by blue arrows. Vị 90kbp We want to use EcoRI to insert gene g (a small piece of DNA) into this cloning vector. The 9i is 20 kbps long. We prepare three (3) samples: a) only cloning vector (v). b) cloning vector (v,) + EcoRI, c) cloning vector (v) + ECORI + gene g1. and run them in a gel electrophoresis. Ethidium bromide (EthBr) is used to stain dsDNAs. If we observe five bands in the last sample (third column) as shown below, please explain what each band represents. Vị only Vị + EcoRI v; + EcoR1 + g; 130 kbp+ 110k:bp 105 kbp- 90 kbp 85 kbpA 2.0kb bacterial plasmid ‘BS1030’ is digested with the restriction endonuclease Sau3A; the plasmid map is depicted in the diagram below and the Sau3A (S) restriction sites are indicated. Which of the following DNA fragments do you expect to see on an agarose gel when you run Sau3A-digested plasmid ‘BS1030’ DNA? a. 250 bp, 450 bp, 550 bp, 1.1 kb, 1.5 kb and 2.0 kb b. 2.0kb c. 250 bp, 400 bp, 450 bp, 500 bp and 550 bp d. 100 bp, 200 bp, 250 bp, 400 bp, 500 bp and 550 bpAfter mutagenesis of wild type Vibrio fisheri, you isolate two different mutant strains (A and B) that, unlike the wild type cells, fail to luminesce when grown to high density in a flask with appropriate medium. Curiously, however, when you inoculate both mutant strains in the same flask, you observe that the mixed (A+B) culture begins to emit light after growing dense. a) What gene/functions are likely affected in each of the two mutants? b) How does this explain their phenotypes?
- Question 1 a) Describe 2 approaches that you can use to determine that you have successfully PCR amplified Sonic Hedgehog. b) What are the key modifications to the PCR primers, SHFw1 and SHRv1 in order for you to clone the PCR amplified Sonic Hedgehog into the multiple cloning site (MCS) of pSELECT-CGFP-blasti. Explain your answer in detail c) After you have successfully cloned Sonic Hedgehog into pSELECT-CCFP-blasti and transfected HEK 293 cells (human embryogenic kidney cells), you observed, using confocal microscopy that the GFP fluorescence displays a reticulate network pattern in the cytoplasm of the cells. Describe an approach you can use to determine the subcellular localization of the Sonic Hedgehog-GFP fusion protein.Genomic DNA from a family where sickle-cell disease is known to be hereditary, is digested with the restriction enzyme MstII and run in a Southern Blot. The blot is hybridised with two different 0.6 kb probes, both probes (indicated in red in the diagram below) are specific for the β-globin gene (indicated as grey arrow on the diagram below). The normal wild-type βA allele contains an MstII restriction site indicated with the asterisk (*) in the diagram below; in the mutated sickle-cell βS allele this restriction site has been lost. What size bands would you expect to see on the Southern blots using probe 1 and probe 2 for an individual with sickle cell disease (have 2 βS alleles)? Probe 1 Probe 2 (a) 0.6kb 0.6kb and 1.2kb (b) 0.6kb and 1.8kb 0.6kb, 1.2kb and 1.8kb (c) 1.2kb 0.6kb (d) 1.8kb 1.8kb a. (a) b. (b) c. (c) d. (d)Taxol is a compound used in cancer treatment. You are working for Genentech on a project to optimize the production of taxol purified from recombinant E. coli bacteria. You have two new strains of SuperGro E. coli: Strain A and Strain B, that you have engineered to express taxol. You want to know which of the two SuperGro E. coli strains is better to use for purifying taxol based on the amount you purify (measured by final concentration of protein in mg/mL). You also want to know which growth media (LB Media or SOC Media) results in a higher amount of purified taxol. You collect data and plot the average final concentration of taxol from each experimental condition in the graph below. Use the approach we discussed in class and write your analysis and interpretation of the data (describe the graph, describe the data, and interpret the data). Make sure to give clear and complete descriptions. A. Describe the graph: B. Describe the data: C. Describe the interpretation:
- The plasmid cloning vector pBR322 is cleaved with the restriction endonuclease PstI. An isolated DNA fragment from a eukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are selected by growth in the presence of tetracycline. The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one end. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below. What does each pattern say about the cloned DNA? Note: pBR322, the PstI and EcoRI restriction sites are about 750 bp apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted.1) You wish to make a restriction map of a 17.0 kb linear fragment. You digest the fragment with Sbf1, Pst1, and a mixture of Sbf1 and Pst1. From these results, you obtain these fragments following agarose gel electrophoresis of the three samples: Sbf1: 7kb and 10kb Pstl: 3kb, 6kb and 8kb Sbf1 and Pstl: 1kb, 2kb, 6kb and 8kb Question: In which of the Pstl fragments is the Sbfl site located? 8kb O 6kb 3kb O None of the abovea)Dr. Thisisaneasyexam decides to amplify a gene from a plasmid using PCR. She starts out with 6.6 x 10-14g of a 10 kb template in a 100 µl reaction. Assuming that the molecular weight of the average base pair is 660 Daltons calculate the number of molecules of the template. b)Given that the concentration of each primer (20 base pairs each) is 0.1µM in this same reaction volume (100 µl) calculate the number of molecules of the primer present
- A piece of DNA 5.0 kb long is cloned and then cut out of the vector for analysis. This linear piece of DNA is digested with two restriction enzymes, EcoRI and BamHI, individually and in combination, and the resulting fragment sizes are determined by electrophoresis. The results are as follows: Restriction fragment size 4.5 kb; 0.5 kb 3.0 kb; 2.0 kb 2.5 kb; 2.0 kb; 0.5 kb Enzyme name EcoRI ВатHI EcoRI + BamHI Construct a potential restriction map based on these results.Question 1 a) What are the key modifications to the PCR primers, SHFw1 and SHRv1 in order for you to clone the PCR amplified Sonic Hedgehog into the multiple cloning site (MCS) of pSELECT-CGFP-blasti. Explain your answer in detail b) After you have successfully cloned Sonic Hedgehog into pSELECT-CCFP-blasti and transfected HEK 293 cells (human embryogenic kidney cells), you observed, using confocal microscopy that the GFP fluorescence displays a reticulate network pattern in the cytoplasm of the cells. Describe an approach you can use to determine the subcellular localization of the Sonic Hedgehog-GFP fusion protein.Ligation is an essential step in the cloning process. It refers to the joining of the gene of interest to the vector using DNA ligase. (i) Determine FOUR (4) control groups which are important to determine the success of this step and also to troubleshoot if any problem should occur. In an experiment, 100 ng vector was added into ligation reaction with 50 ng of insert (500 bp). The desired vector: insert ratio of 1: 3 was used. Determine the size of the (ii) vector.