School of Engineering and Science - Jacobs University

Material and Methods
dissolved in the buffer by 10 min incubation at 50°C in a thermomixer. During
this incubation the sample was mixed every two minutes. After complete
disolvance of the agarose piece, column purification was performed in
accordance to the manual. The DNA was eluted in water and stored at –20°C.
4.2.5.6 QIAquick PCR and reaction purification kit
The QIAquick-kit (Qiagen, Hilden) was used to re-isolate DNA out of reaction
mixtures. 5 volumes of buffer PB were added to the reaction. After that the
mixture was loaded to a QIAquick spin column in a 2 ml collection tube by short
centrifugation. Then the column was washed with 0.75 ml of buffer PE and
placed into a 1.5 ml plastic reaction tube. To elute, 30 µl of sterile water was
added to the center of the column and incubated at room temperature for
1-3 minutes. The sample was shortly centrifuged (1 min, 13,000 rpm). DNA
samples were stored at –20°C.
4.2.5.7 Estimation of DNA concentration
Concentration of DNA samples was determined photometrically, as an
absorption of 1 at λ = 260nm correlates to approx. 50 µg DNA per ml solution
(Sambrook et al., 1989).
4.2.5.8 Ligation of DNA
T4 DNA ligase catalyses the formation of phosphodiester bonds at juxtaposed
5’-phosphate and 3’-hydroxyl ends in duplex DNA. The appropriate insertion of
a piece of insert into an opened vector by this reaction is most efficient, if a
relatively small amount of vector (20-100 ng) is mixed with an 3-8 fold excess of
insert DNA. To high concentration of vector facilitates self-ligation of vector
DNA while to low concentration of insert DNA minimizes ligation efficiency. The
ligation reaction contained 1 µl of 10x buffer and 1 µl of T4 DNA ligase. It was
incubated at 18°C for about 20 hours. Subsequently the ligated DNA was
transformed into E. coli DH5α. The ligation reaction was stored at –20°C.
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