Question

TTQ: You’ve just isolated a 2.2 Kb PCR product that contains the Unicorn gene. You’ve engineered in a PstI site at each end of the fragment.

•Through pst Idigest and ligation, you clone the Unicorn gene into the MCS of a cloning vector that is 4 Kb.

•You get several colonies that grow on Amp and you isolate the cloning vectors from several different clones.

Draw what the gel would look like if you cut with PSt1 for a:

•Transformed (T1) colony with the plasmid and Unicorn gene successfully inserted after digestion with Pst I

•Transformed (T2) colony with the plasmid and no Unicorn gene after digestion with Pst I

•What a non-transformed (NT) colony that has no plasmid would look like.

Would someone mind explaining this in words as well? I'm really confused on how to do it. Thanks!

Answer 1

1) if the plasmid is recombinant then if we digest with PstI, we will get 2 bands, one corresponds to the inserted gene of size 2.2 Kb and one corresponds to the plasmid which is 4 kb, when we digest the non-recombinant plasmid, PstI will cut at the MCS and produce a linear DNA of size 4 kb and in the non-transformed cell, there is no plasmid ( assuming there are no other plasmids, for the restriction digestion we isolate only plasmid and not the genomic DNA) so there will be no bands.

( we have used PstI for cloning so the Insert has 2 sites on 2 ends and the vector has only one site to where the gene is inserted so after ligation there will be 2 sites for the PstI)