title: Whitehead Mouse 13K Promoter Array
status: Public on Jan 31 2007
contact.city: Seatttle
contact.first: Yi
contact.last: Cao
contact.email: ycao@fhcrc.org
contact.state: WA
contact.address: 1100 Fairview Ave. N
contact.department: Human Biology
contact.country: USA
contact.institute: Fred Hutchinson CRC
contact.phone: 206-667-5278
organism: Mus musculus
description: To prepare the DNA content of the arrays, the program Primer3 was used to design primers using the sequences described above. PCRs were performed on these primer sets using standard conditions, except for the presence of 1 M betaine in all PCR reactions. Betaine was empirically observed to increase the success rate of the amplification reactions. Of the 13,000 pcr pairs, 70% gave a strong band of the appropriate size, as verified on 2% agarose gels. We have noted, however, that PCR products undetectable by agarose EtBr gel analysis can give valid positive signals when concentrated and printed on the DNA arrays. PCR quality evaluations were performed on the BRIDNA suite of programs from the Biotechnology Research Institute of the National Research Council of Canada. PCR products were recovered from the reaction mixture by ammonium acetate/isopropanol precipitation and resuspended into 3x SSC with 1.5 M betaine to minimize evaporation and improve spot quality. The resuspended DNA was transfered to 384 well plates and printed on GAPS-coated slides (Corning) using a robot from Cartesian Technologies. The quality of the arrays was determined on a batch-wise basis by hybridization with sequence-neutral oligonucleotides covalently linked to Cy3 or Cy5, followed by calculation of usable percentage of spots, combined with direct visual inspection of the quality of the chip. Because the genome was only approximately 70% complete in the April 2001 alignment used to generate the primer sets for this array, the Hu13K array was remapped against the August 2003 final release of the completed human genome post-production.
manufacture_protocol: As in http://www.wi.mit.edu/young/research/genome/protocols.html