Oligonucleotide synthesis was carried out both, by a PCR-MATE EP391 DNA synthesizer, and a commercial manufacturer (MWG-Biolabs). The commercial product was delivered ready to use, whereas the non-commercial ones were delivered as crude elution products in concentrated ammonia. The protecting groups were cleaved off by overnight incubation at 55psy176 C. A vacuum evaporation of ammonia and ethanol precipitation (see section EtOH) was performed to purify short primers, whereas longer oligonucleotides were purified by Oligonucleotide Purification Cartridges (Applied Biosystems) using the following protocol:
The cartridge was flushed with 5 ml acetonitrile, followed by 5 ml 2.0M
triethyl amine acetate. About 50 to 100 OD units of the crude, deprotected
oligonucleotide still in concentrated ammonia were diluted with one third
volume of bidistilled water to give a final volume of 4 ml. This solution was
slowly pushed through the cartridge. The eluted fraction was saved and pushed
through the cartridge once again. The loaded cartridge was slowly washed with 5
ml 1.5M ammonia hydroxide three times, and flushed with 5 ml water two times.
The oligonucleotide was now detritylated with 5 ml of 2% trifluoroacetic
acid, flushed with 2 
5 ml water again and the purified, detritylated
oligonucleotide eluted by slowly washing the cartridge with 1 ml 20%
acetonitrile.