Details of Microarray construction (primer design/PCR/purification/spotting)
| The primary goal of our microarray efforts is to array DNA sequences obtained from the Antarctic picoplankton as "target" DNA on microarrays, then to perform hybridization experiments with mRNA isolated from samples collected during our field seasons in order to study expression of genes in the natural samples and to optimize steps where necessary. |
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.
