Answer to Question #93332 in Molecular Biology for Kylie

Question

3.1 Describe the process of cloning a DNA fragment into the PstI site of the vector pBR322.
How would you screen for clones that contain an insert?

Answer 1

At first, the DNA fragment and the vector pBR322 must be digested with the restriction enzyme PstI. Next, the digested fragment and the vector must be resolved in the agarose gel and purified from the gel using the appropriate kit or procedure. The vector should be dephosphorylated using alkaline phosphatase. Then, the fragment and the vector are ligated using ligase; competent E.coli cells are transformed with the ligation mixture and plated on LB agar. The screening of the colonies should be performed using the same restriction enzyme. The colonies are transferred to LB medium and grown overnight. The next day, plasmid DNA is extracted from the bacteria suspension. The purified DNA is then digested with PstI and resolved in agarose gel. The presence of the DNA fragment of the appropriate size will indicate the presence of the insert.