Sticky to Blunt end (Hypothetical) - (Sep/27/2013 )
I have a hypothetical question. If I use a restriction enzyme, such as HinDIII on a plasmid vector to create sticky ends, is there a way to convert the overhangs to blunt ends without using Klenow fragment? There are restriction enzymes from NEB which recognize sequences of A and T only which would cleave the 3' overhang (like HpaI). Would this even work, or are my fundamentals lacking?
This would not work. The restriction enzymes recognize the helical form of double stranded DNA with specific bases. After being cut, the free end of the DNA strand no longer has this conformation. In general, restriction enzymes do not work well close to the end of DNA strands -- some are more sensitive than others.
you can use T4 DNA polymerase or Mung bean nuclease to make blunt end