Ligations of PCR product into pGEM T Vector

Vector is available from Promega (catalog # A3600). Vector sequence file in Genbank format is available here.

TA Cloning into the T Vector system relies on the Taq polymerase adding an A overhang to the PCR product. However, not all Taq polymerases do so. Therefore, before using the system please verify that the Taq polymerase you are using does NOT have 3' to 5' exonuclease activity (this should be included in the description of the Taq). If the polymerase does have extensive 3'-5' exonuclease activity (ie produces blunt ends) you can add A overhangs by incubating the PCR product with Taq (though, pragmatically, this normally is more trouble than what it's worth...)

Protocol:

Ligation

5µl 2x ligation buffer

0.5µl T plasmid DNA

2-3.5µl PCR product (depending on band intensity)

1µl T4 ligase (from the kit)

O/N in the fridge

Transformation

T plasmid can be used for blue-white selection. To identify clones with insertions, spread 20-50µl Xgal on the plate, and let dry for 30 minutes before you spread your culture. (IPTG is not needed for induction, since pGEM T is a high copy number plasmid.)

The next day, select white colonies that are in the proximity to blue colonies (since Xgal with be unevenly distributed on the plate). I generally use colony PCR for screening, using primers 1K15+1K16 (you can also use T7+M13reverse).