Question

Suppose you have a DNA fragment you would like to insert into the pSAP plasmid. The fragment has PstI and EcoRI restriction endonuclease sites near the 5' end and HindIII and Smal restriction endonuclease sites near the 3' end. Choose the best restriction endonuclease(s) to digest the both the DNA fragment and pSAP. Thank you for all the help everyone!!! :)

Answer 1

To insert a specific DNA fragment into the pSAP plasmid, EcoRI and HindIII are suitable restriction endonucleases to use if these sites are available on the plasmid. They create sticky ends that facilitate the ligation step, leading to the formation of a recombinant plasmid.

Step-by-step explanation:

To insert a DNA fragment with PstI and EcoRI sites near the 5' end and HindIII and Smal sites near the 3' end into the pSAP plasmid, you should select restriction endonucleases that create complementary sticky ends on both the fragment and the plasmid. In this case, it's critical to choose endonucleases that are also present in the multiple cloning site (MCS) of the pSAP plasmid. Often, EcoRI and HindIII are used because they create sticky ends compatible for DNA ligation, so these enzymes are promising candidates for the plasmid digestion as well, given that they are part of the MCS of the plasmid. After digestion, DNA ligase can be used to join the sticky ends and create the recombinant plasmid.

The whole procedure simplifies to choosing the enzymes, cutting the plasmid and the insert with these enzymes, and then using DNA ligase to merge them into one continuous DNA molecule which would then contain the fragment of interest within the plasmid backbone.