2. You want to express the Purple gene in bacteria. This is a mouse gene respons

Question

2. You want to express the Purple gene in bacteria. This is a mouse gene responsible for bright purple streaks commonly observed in the hair of some mouse species. You have the cloned Purple gene, with its natural promoter, and also a bacterial expression vector. Restriction maps for both of these are shown below. Describe a strategy for cloning the Purple gene into the expression vector 2 Am Ori Purple gene promoter Bacterial promoter Dpnl Xhol EcoRI Ori urple CA TATG Amp GT/ ATAC CT AG Dpnl: GA/TC CAGC BamHl: G/GATCC Notl: GC/GGCCGC Clal: AT/CGAT Pstl: CTGCA/G EcoRv: GAA de usin

Explanation / Answer

1. digest the gene with Dpn1 and Sma 1. both are blunt end producing RE.

2. digest the vector with Dpn1 and Eco RV enzyme. (Dpn1 site is present inside Bam H1 RE site).

3. now mix both the gene insert and vector and perform blunt end ligation reaction.

4. your gene will be in frame with the bacterial promoter.

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