Bio-Rad Ligation and Transformation Module User Manual

Page 21

DNA ligation with blunt ends — Blunt-end ligation, in which both the
inserted DNA and the vector have blunt ends, has an advantage compared
to sticky-end ligation in that all DNA ends are compatible with all other
ends. In other words, it is not necessary to cut the vector and insert with
the same restriction enzymes to get complementary overhangs as for
sticky-end ligation. Vectors used for blunt-end ligation have a blunt-ended
ligation site in the MCS. They still have an MCS, as the restriction enzyme
sites are very useful for subsequent manipulation of the inserted DNA.

LIgation of PCR fragment into vector.

In PCR,

Taq DNA polymerase adds a single nucleotide to the 3'-end of the

PCR product, usually an A. Since this A overhang would prevent blunt-end
ligation, it must be removed prior to ligation. Treating the PCR product with
a proofreading DNA polymerase removes the 3'-A, leaving blunt ends
ready for ligation. (Not all thermally stable DNA polymerases used in PCR
leave an A overhang; some polymerases like

Pfu DNA polymerase have

proofreading ability.)

Features of the pJet1.2 blunted vector

The pJet1.2 blunted vector has several features that make it a good choice
for this laboratory activity:

•

It is a vector designed for blunt-end cloning and is already linearized
with blunt ends

PCR Fragment