By use of a peltier thermal element, the immobilised products kept in suspension were heated in deionised water to 80°C for one second and cooled to room temperature.
Quantification of DNA was carried out using Nanodrop ‘ND1000′ spectrophotometer. Besides, the biotinylated amplification products were bound to Dynabeads M 270″ Streptavidin beads during an incubation at room temperature using a highsalt binding buffer and when bound, washed with 1 × TE buffer. Elution was achieved by breaking streptavidinbiotin bond in a ’20 μl’ volume using deionised H2O. Efficient elution is achieved through a combination of elevated temperature, incubation or even appropriate temperature ramping at the elevated temperature, as described in more detail elsewhere. As a result, during incubation the beads were kept in suspension by mixing through pipetting almost any third minute. I’m sure you heard about this. That’s right! Beads were separated from released products by magnetic separation, reconditioned through a repeated wash procedure with 1 × TEbuffer prepared, decisively and for purification next round.
We exemplify the method performance by studying 4 purification representative biotinylated amplification products and subsequently show that arrays prepared using this method will successfully be used for largescale transcriptional profiling.
This work was supported by grants from the Wallenberg Consortium Alice, Knut and North Wallenberg foundation and Swedish Scientific Research Council. Just think for a moment. The purification method is on the basis of reversible biotinstreptavidin binding, utilises streptavidincoated paramagnetic beads and will be automated on a robotic workstation dedicated for magnetic separation and equipped with a temperature control. In this study we present a method suitable for purification of gene sequence tags, that have lately been designed and successfully used for transcriptional plant profiling model system Arabidopsis thaliana.
We present an alternative solidphase purification strategy suitable for efficient preparation of pretty biotinylated, extremely or rather short specific probes suitable for vast scale expression profiling.
Arrays use has been exemplified by analysis of gene expression overlooking caused by a 3 hour indole3acetic treatment. With that said, twenty one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. In any event, we present an alternative solidphase purification strategy suitable for efficient preparation of fairly biotinylated, extremely and shorter specific probes suitable for massive scale expression profiling. Normally, the arrays use has usually been exemplified by analysis of gene expression overlooking caused by a 3 hour indole3acetic treatment. Twenty one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology.