Problem:
You want to insert a DNA fragment into pT5 plasmid . The DNA fragment is an open reading frame with ATG on the 5’end. pT5 plasmid contains a bacterial promoter at 0.8 kb position, EcoRI site at 0/6 kb, HindIII site at 1 kb, BamHI site at 3 kb. ORF is a HindIII fragment 2 kb long with EcoRI site at 1 kb and BamHI site at 1.5 kb.
Required:
Question 1: What will be the right orientation of a fragment in the plasmid and which enzyme you can use to find out the orientation of the ORF after ligation?
Question 2: Show the sizes of the fragments that will be produced after restriction digest with the chosen enzyme. Explain your answer.
Show your calculations, but you do not need to write out any explanation.