Many DNA sequence manipulations are made easier by cloning a PCR
product into an appropriate vector. This allows large-scale
replication of DNA in bacteria, DNA sequence confirmation and
addition of restriction endonuclease sites for easy subcloning
of the target DNA. Standard PCR with Taq DNA polymerase produces
PCR products with a single deoxyadenosine overhang at each 3´
end. These single-nucleotide overhangs anneal to the
3´‑thymidine overhangs of T-cloning vectors such as the Promega
pGEM®-T (Cat.# A3600) and pGEM®-T Easy Vectors (Cat.# A1360) and
the Invitrogen TOPO® TA Cloning Kit with pCR®2.1-TOPO® Vector
(Invitrogen Cat.# K4500-01). Theoretically, smaller inserts will
be cloned more efficiently than longer inserts. We tested the
effect of PCR product length using three T-cloning systems. PCR
cloning efficiency was determined using PCR products ranging
from 542 to 3,100 base pairs with the pGEM®-T and pGEM®-T Easy
Vectors and pCR®2.1-TOPO® Vector. Four different inserts were
amplified and cloned into