Hi there, any help on this would be great :) 1. Obtain a map of pGEM-3Z with uni

Question

Hi there, any help on this would be great :) 1. Obtain a map of pGEM-3Z with unique restriction sites shown. One source is the link: http://www.snapgene.com/resources/plasmid_files/basic_cloning_vectors/pGEM-3Z/. From the map, consider the unique sites AlwNI, TsoI, and KpnI. Which of these would be suitable/unsuitable for a subcloning experiment in which you wished to introduce a gene into pGEM-3Z? Give reasons for your answers. [3 marks] [Clue: Check the locations of the sites and any effects introducing inserts might have on the plasmid/experiment] Thank you.

Explanation / Answer

pGEM-3Z is a 2743bp plasmid. It contains an OriC , a ampicillin gene and a lacZa gene. It is a suitable shuttle vector containing many restriction sites.

AlwNI site at 861 lies in the replication origin region of plasmid, thus its is unsuitable because if we use it plasmid won’t be able to replicate in the host.

Tsol site lies at 1737 in the ampicillin resistance gene. Use of this gene does not provide us benefit as selection of recombinants can be a challenge without ampicillin gene selection marker.

Kpnl site is located in lacZ gene at 21 position. Kpnl produces sticky ends which are perfect for cloning purposes. Insertion at Kpnl site will cause disruption of LacZ gene. Which can be employed for screening of successful transformants. Blue white screening can be used to check successful ligation of insert in plasmid.

Therefore, Kpnl would be the best choice .

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