Question

The following questions are based on the plasmid maps outlined below. GFP (700 bp) Plasmid B (5.7 kb) ECORI Kpnl BamHI - Xmal Plasmid A (4.2 kb) Ampicillin resistance Kanamycin resistance 15. Outline the strategy for how you would clone the GFP cassette from plasmid A into plasmid B? Please include descriptions of how you would select for potentially correct clones and verify the final construct. For the purposes of this exercise, assume that sequencing services are not available. * Your answer 16. If both plasmids contained the same antibiotic resistance cassette, what additional steps would you take to assess whether your cloning strategy was successful? * joux IIPUIH- Notl Agel Ascl Promoter Terminator

Answer 1

15) first culture the cells having the plasmid A, if we don`t have it transform the E.coli cells with plasmid A and grow those cells in kanamycin plates, so as to select the transformed cells, culture those cells, and isolate the plasmid from those cells so as to get a large amount of the plasmid then digest the plasmid with Age I and XmaI, run the digested product in the agarose gel. then elute the small band which contains the GFP.

then digest the plasmid B with Age I and Xma I and ligate the plasmid B with small band eluted, using DNA ligase to get the recombinant DNA. then transform the cells with the recombinant DNA.

plasmid B has the ampicillin resistance gene so the cells transformed with the recombinant plasmid can be selected using the ampicillin plates.

culture the colonies that grow in the ampicillin plates and isolate the recombinant plasmid.

16) if both plasmids have the same antibiotic resistance gene, then we have to do restriction digestions to check the recombinant DNA. AscI site is present only in the GFP gene, so digest the isolated plasmid with AscI and BxtI if the GFP gene is incorporated into the plasmid B we should get 2 bands, BxtI site is present only in the plasmid B and AscI site is found only in the GFP, if the BxtI and AscI combined digest produces 2 bands, one small and one large band then the recombinant DNA was created successfully also the transformation was successful.