PMC:5003436 / 11920-14268
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/5003436","sourcedb":"PMC","sourceid":"5003436","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5003436","text":"2.4. Microarray Design\nProbe design was done with the open software package ARB [18]. All oligonucleotides including the positive and negative controls were synthesized by Thermo Fisher Scientific (Ulm, Germany) with a C6 aminolink at the 5' end of the molecule. The probes had a length between 18 and 25 nt and a 15 nT-long poly (dT) tail following the NH2 link at the 5' end. Table 2 shows a list of the probes and their targets. The complete hierarchy for each probe can be found in the GPR-Analyzer which is available online at http://folk.uio.no/edvardse/gpranalyzer. The probe sequences are patent pending and a commercial kit will soon be available from Kreatech containing the array and all reagents for hybridization. Epoxy-coated slides (Genetix or Schott) of MIDTAL version 3.2 were printed using a pin printer VersArray ChipWriter Pro (Bio-Rad Laboratories GmbH, Munich, Germany) and split pins (Point Technologies, Inc., CO) as described by Kegel et al. [16]. One array contained 136 different probes and 4–8 replicates, as well as three negative (NEGATIVE1_dT, NEGATIVE2_dT, NEGATIVE3_dT), one positive control (TBP = TATA-box binding protein), Poly-T-Cy5 (spotting control), and two internal controls (DunGS02_25_dT and DunGS05_25_dT for Dunaliella tertiolecta) (MIDTAL ver3.2). After spotting, slides were incubated for 30 min at 37 °C and then stored at −20 °C.\nPrinting of MIDTAL slides version 3.3 was done by Scienion AG using a sciFlexarrayer S11 and epoxy-coated slides from Genetix. One array contained eight replicates of 140 different probes including the seven controls stated above. After printing, the slides were transferred to a 75% humidity chamber, kept there overnight at RT, and stored afterwards in a sealed aluminum bag refilled with argon at 4 °C.\nmicroarrays-02-00001-t002_Table 2 Table 2 Summary of probes designed or modified from published FISH probes and used to form the third generation of the MIDTAL microarray, including the targeted species, and whether it was made from the 18S or 28S rRNA gene. Probe sequences are not provided because the microarray is patent pending and will soon be commercially available from Kreatech, Amsterdam, The Netherlands. A complete taxonomic ordering of the probes can be seen in the GPR-Analyzer program and the MIDTAL hierarchy file that comes with that program.\n\n2","divisions":[{"label":"Title","span":{"begin":0,"end":22}},{"label":"Table caption","span":{"begin":1785,"end":2347}}],"tracks":[]}