Following transformation with the CRISPR plasmid, the yeast will be plated on SD-URA plates. Why is SD-URA media used? Name the relevant marker gene in the pCRCT CRISPR plasmid.
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3A. Following transformation with the CRISPR plasmid, the yeast will be plated on SD-URA plates. Why is SD-URA media used? Name the relevant marker gene in the pCRCT CRISPR plasmid.
3B. What would you expect to see if you plated on YED accidentally? Will most of the yeast be red or white? Why?
3C. The complete genotype of the host yeast strain is MATa ade2 his3 leu2 met15 ura3. Name one advantage of using a yeast strain with auxotrophies for several genes (i.e. his3, leu2 and ura3).
3D. After successful CRIPSR editing, the yeast can later be “cured” of the recombinant CRISPR plasmid – that is, the plasmid is lost but the CRISPR edit is stably inherited. Write the new genotype of the yeast-based on the ADE6 gene.
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- 1a.The complete genotype of the host yeast strain is MATa ade2 his3 leu2 met15 ura3. Name one advantage of using a yeast strain with auxotrophies for several genes (i.e. his3, leu2 and ura3). b.)After successful CRIPSR editing, the yeast can later be “cured” of the recombinant CRISPR plasmid – that is, the plasmid is lost but the CRISPR edit is stably inherited. Write the new genotype of the yeast based on ADE6 gene.Part A. If student counts 63 colonies on their 10^-6 dilution LB plate. What was the original concentration of their cells if they plated 100ul? Part B.If we used pGFPuv as the template for PCR positive control. This is because: a. it contains the GFP gene so it should show a product. b. It contains DNA fragments that were added to the ligation reaction. c. It is the desired plasmid we wanted to make. d. So we have a band to compare our unknown plasid to allowing us to check if the unknown is the right size.7. Consider the following plasmid (size 6700 bp), with restriction sites at the positions indicated: BamHI + 1 6700 bp 2800 3500 EcoRI BamHI Probe
- A target gene for producing human insulin must be inserted into a plasmid before the transformation process. Why the plasmid is important in this process? * I The target gene lacks the ability to replicate itself. II Screening process will be difficult without cloning vector. II The host cell is able to accept the recombinant plasmid but not the non- recombinant plasmid. O I and Il only I and III only O Il and III only O I, Il and III1. Determine what is being meant by the statements: a. What is a binary vector? What characteristic does it have? b. What is the role of the vir genes in the Ti plasmid c. System for negative regulation of transcription of bacterial genes involved in lactose metabolism.7) Why are the following reagents used? Neutralizing solution (Plasmid isolation) Isopropanol (Plasmid isolation) RNase (isolation of genomic DNA)
- Comment on the failure of p1vir transduction experiments and give a reason why you think the transductions was unsuccessful for some genes. Suggest any alternative methods that could be used to create the mutations that were unsuccessful in these experiments? There are no colonies on the plate for some of the genes. and in DNA sequence, some colonies don't have primers present. For PCR, some colonies are not replaced by kanamycin or tetracycline.1. As described in the textbook, the plasmid vector pBluescript II contains an ampicillin resistance gene and a lacZ gene, within which is located a multiple cloning site. Briefly describe how one selects for/identifies bacterial cells containing recombinant pBluescript II molecules and explain how the selection/identification works.A B Figure 1 The postgraduate student, Vanessa, cloned her gene of interest into two different vectors; pUC18 and pZERO®-1, and transformed the competent cells with the recombinant vectors. After that, she plated them on the antibiotic plates and incubated her samples for 18 hours. The next day, she realized that the plates were not labelled. (i) Based on the observation above, can you identify which plate is carrying the recombinant pUC18 and pZErOⓇ-1, respectively? (ii) Why are there small colonies surrounding the big colonies in Figure 1 (A)? Explain your answer in detail. (iii) Do you think all the colonies present on the plates in Figure 1 (B) contain the gene of interest? Describe the selection procedures for recombinant pZErO®-1.
- A.How could endonucleases interfere with the transformation procedure? B. Does supercoiled or nicked plasmid get transformed more efficiently? Why?1. You have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions. a. PscI & GsuI b. ScaI, PdmI & BsaXI c. ScaI, SspI & EheI 2. You have determined that the amplicon you want to clone has enzyme restriction sites for HindIII and EcoRI. After investigation you have seen that the pUC18/19 plasmid also have these enzyme restriction sites in its multiple cloning site (MCS on map above). After enzyme digestion your amplicon is 854 bp long. a. What length will the recombinant plasmid be after you have inserted your amplicon? Show your calculation. b. In the amplicon insert you have an enzyme restriction site for NdeI at 500 bp. If you digest the recombinant plasmid with this enzyme what length will the fragments be?A cloning vector map is shown below. EcoRI Bam Ban Hind P-galactosidase Amp Bam Bam EcoRI Ori C Which restriction site is best for inserting a DNA fragment for selection of chimeric plasmid containing colonies? 1) They're all equally good. 2) Hindll 3) EcoRI 4) BamHI