School of Engineering and Science - Jacobs University

Material and Methods
4.2.5.9 Ligation of PCR fragments by T/A cloning
One attribute of the Taq polymerase is the creation of a so-called A-overhang.
After finishing the polymerisation of the according PCR fragment, a terminal
adenine is attached to the fragment. The pGEM-T Easy cloning kit provided by
Promega uses this feature for an enhanced cloning of PCR fragments. The kit
provides linearized DNA of the pGEM-T Easy vector extended by a terminal
thymine. The combination of T- and A-overhang provide a sticky end that highly
increases ligation efficacy.
For pGEM-T Easy cloning the purified PCR fragment was mixed with 0.5 µl of
vector solution, 2µl of 5x rapid ligation buffer and 5 U of high concentrated T4
ligase in a 10 µl reaction mixture. The ligation was incubated at 18°C over night
and subsequently transformed into E. coli DH5α. Ligation reaction was stored
at –20°C.
4.2.5.10 Preparation of electrocompetent E. coli cells
Electrocompetent cells of E. coli DH5α were prepare according to the standard
protocol (Sambrook et al., 1989). A fresh over night culture of DH5α was
inoculates at a dilution of 1:100 and incubated at 37°C until exponential growth
phase was reached (OD 600nm = 0.5 - 0.8). The cells were incubated on ice for
30 min. and then harvested by centrifugation (600rpm; 4°C). Next, medium
residues and salts were removed from the cells by three subsequent washing
steps (two times ice cold water, one ice cold 10% glycerol). The resuspension
volume was reduced in each step and after the final washing step the cells
were resuspended in 1/500 volume of the initial culture volume. 40 µl aliquots
of electrocompetent cells were filled into 0.5 ml plastic reaction tubes and
directly shock frozen in liquid nitrogen. The cells were stored at –80°C.
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