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Posted: **Wed May 18, 2022 7:27 pm**

: Your Plan To Clone Your 400 Bp Pcr Product Generated In Section B From The Wt Hfe Gene Into The Plasmid Pgreen Illustrat 1 (54.43 KiB) Viewed 38 times

: Your Plan To Clone Your 400 Bp Pcr Product Generated In Section B From The Wt Hfe Gene Into The Plasmid Pgreen Illustrat 2 (19.28 KiB) Viewed 38 times

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 cut site positions as indicated on the diagram Noti (1) EcoRV (3500) ori Kans p Amp KpnI (900) PGREEN 4000 bp P GFP Pvul (2200) Bg/I! (1800)
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 4? Motivate your answer [2]