You prepare a linear PGREEN plasmid by digestion with the enzyme identified in (3) above, ligate it to the PCR product,
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You prepare a linear PGREEN plasmid by digestion with the enzyme identified in (3) above, ligate it to the PCR product, and transform it into bacteria. What phenotype wold you expect for bacteria that have taken up recombinant plasmids, versus those with non-recombinant plasmids? [3] 5. With a non-directional cloning such as this, recombinant plasmids can contain an Insert ligated Into the vector in two different orientations. Explain how a double digest with Noti and Rsal will allow you to distinguish between these two types of recombinants. [2] 6. Will a bacterium containing a recombinant plasmid be able to the express the part of the human protein encoded by the HFE gene exon 4Motivate your answer [2]
You prepare a linear PGREEN plasmid by digestion with the enzyme identified in (3) above, ligate it to the PCR product, and transform it into bacteria. What phenotype wold you expect for bacteria that have taken up recombinant plasmids, versus those with non-recombinant plasmids? [3] 5. With a non-directional cloning such as this, recombinant plasmids can contain an Insert ligated Into the vector in two different orientations. Explain how a double digest with Noti and Rsal will allow you to distinguish between these two types of recombinants. [2] 6. Will a bacterium containing a recombinant plasmid be able to the express the part of the human protein encoded by the HFE gene exon 4Motivate your answer [2]
Video ald: Plasmid Cloning Your plan to clone your 400 bp PCR product generated in Section B from the WT HFE gene into the plasmid PGREEN Illustrated below. The PCR amplicon has been enzymatically treated to ensure it is fully blunt-ended, and purified from the PCR reaction mixture. PGREEN (4000 bp) contains the GFP (green fluorescent protein) gene, which upon expression in a bacterial hosts results in green colonies. It also contains two antibiotic resistance genes, ampicillin resistance (amp") and kanamycin resistance (kan") and a number of unique restriction enzyme (RE) sites with aut site positions as indicated on the diagram Noci (1) BOORV (3500) or Kan Kpni (900) a. PGREEN 4000 bp GFP Pvul (2200) BOVI (1800) 1. Explain the function of the promoter sequences (P) present on the plasmid. [1 ] 2. Explain why ampicillin acts as an antibiotic, and the mechanism whereby the ampl gene functions in bacteria. [2] 3 Which RE site on PGREEN will be the most suitable for cloning of your PCR amplicon? Motivate your answer. [2] 4. You prepare a linear PGREEN plasmid by digestion with the enzyme identified in (3) above, ligate it to the PCR product, and transform it into bacteria. What phenotype wold you expect for bacteria that have taken up recombinant plasmids, versus those with non-recombinant plasmids? [3] 5. With a non-directional cloning such as this, recombinant plasmids can contain an insert ligated into the vector in two different orientations. Explain how a double digest with NotI and Rsal will allow you to distinguish between these two types of recombinants. [2] 6. Will a bacterium containing a recombinant plasmid be able to the express the part of the human protein encoded by the HFE gene exon 42 Motivate your answer
Vidov mld: Plasmid Cloning Your plan to clone your 400 bp PCR product generated In Section B from the WT HFE gene into the plasmid PGREEN Illustrated below. The PCR amplioon has been enzymatically treated to ensure it is fully blunt-ended, and purifled from the PCR reaction mixture. PGREEN (4000 bp) contains the GFP (green fluorescent protein) gene, which upon expression in a bacterlal hosts results in green colonies. It also contains two antibiotic resistance genes, ampicillin resistance (amp) and kanamycin resistance (kar) and a number of unique restriction enzyme (RE) sites with aut site positions as indicated on the diagram Nodt (1) BORM (B500) or Kane Ammo Kpni (900) PGREEN 4000 bp GEP Pvul (2200) BOVI (1800) 1. Explain the function of the promoter sequences (P) present on the plasmid. 2. Explain why ampicillin acts as an antibiotic, and the mechanism whereby the ampl gene functions in bacteria. [2] 13. Which RE site on PGREEN will be the most suitable for cloning of your PCR amplicon? Motivate your answer. [2] 4. You prepare a linear PGREEN plasmid by digestion with the enzyme identified in (3) above, ligate it to the PCR product, and transform it into bacteria. What phenotype wold you expect for bacteria that have taken up recombinant plasmids, versus those with non-recombinant plasmids? [3] 5. With a non-directional cloning such as this, recombinant plasmids can contain an insert ligated into the vector in two different orientations. Explain how a double digest with Notl and Rsal will allow you to distinguish between these two types of recombinants. [2] contact 6. Will a bacterium containing a recombinant plasmid be able to the express the part of the human protein encoded by the HFE gene exon 4? Motivate your answer [2]