Details of Microarray construction (primer design/PCR/purification/spotting)
The project flow schema is found here.
Primer design: Primers were designed to amplify genes identified in Antarctic archaeal contigs. Amplicons were subsets of the full length ORFs between 200-500 bases long. Primers were designed to have uniform Td's, and be between 17-21 bases long.
A program created by Craig Cummings and Colleagues, Design MA was used for design of primers. If interested in this program, contact Craig at cummings@pmgm2.Stanford.EDU.
Primer3 was used for ORFs that Design MA had trouble with using our design constraints.
PCR: Using typical thermal cycling conditions, most ORFs were amplified at 55 for 30 cycles. Two PCR reactions of 100ul were preformed for each ORF and combined for a total volume of 200ul.
Purification: PCR products were purified using a 96-well Qiagen PCR-cleanup columns on a Qiagen 3000 BioRobot liquid handling system, operated at the Nevada Genomics Center. PCR products were dried before resuspension in 1X ArrayIt spotting buffer.
Spotting: PCR products were transferred to 384-well plates for spotting onto TeleChem SuperAmine slides at the Nevada Genomics Center using a humidity controlled Virtek SDDC2 Microarrayer.