DNA into the cut plasmids. Finally, you transform the E. coli bacterial cells wi

Question

DNA into the cut plasmids. Finally, you transform the E. coli bacterial cells with the ligation mix (the recombinant plasmids) Note: The recognition sites for Kpn 1 and EcoRI on plasmi are 1 kb apart EcoR BamH1 Sma I Kpn 1 Sma l promoter BamH2 Kpn 1 BamH1 EcoRi Tet (tetracyclin resistance Amp resistance) o.Skb 15 kb ORI (bacterial origin of replication) 1. Which restriction enzyme (Kpn I, BamH1, EcoRl or Sma I) did you use to digest Gene A for insertion in to the plasmid? 2. Which restriction enzyme (Kpn I, BamH1, EcoR I or Sma I) did you use to digest the plasmid before insertion of Gene A? Briefly explain why.

Explanation / Answer

1) to digest gene A Kpn I is used. because at the both the sides of gene A Kpn I restriction sites are there. a gne which has to be cut and recombined with the vector, must be cut at either the ends of the donor DNA by the same restriction enzyme.

2) to digest the plasmid DNA Kpn I restriction enzyme only should be used. because a donor DNA can be ligated with the plasmid DNA, if it has been cut with the same restriction enzyme, with which the donor DNA has been cut to take out the piece of DNA that is used for recombination.

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