Start Date: Sep 21, 2006
End Date: Dec 31, 2010
Microsatellite library creation. Total genomic DNA is extracted from 2.0 g fresh leaf tissue of target plant using a modified Dellaporta et al. (1983) protocol. The genomic DNA is enriched with the following synthetic biotinylated oligonucleotide probes: (all labeled with biotin-ATAGAATAT at the 5' end): (CT)15, (GT)15, (CAA)10, (ATT)10, (GCC)10, (ACC)10, (AGG)10, (CTT)10, (ACG)10, (AGC)10, (ACT)10, (ATC)10. Two ug of each genomic DNA are digested in 10X reaction buffer (2.5 ul), 1 ul Sau 3Al (10U/ul) and dH20 to final volume of 25.0 ul. The mix is incubated at 37C overnight. The enzyme is inactivated by heating at 65C for 10 minutes. A 500 ng sample of the DNA is electrophoresed on a 0.9% agarose gel and visualized with ethidium bromide to make sure that the DNA has been cut by the restriction enzymes before ligating with the linkers. 200 ng of digested DNA is mised with 4 ul, 10X BSA, 4 ul 10X ligase buffer, 2 ul T4 DNA ligase (Life Technologies, Inc.), ~1 ug Sau 3 AI Linker, and dH20 to a total volume of 40 ul. The reaction is incubated at 4C for 24-66 hours. The ligation is halted by heating the mix at 65C for 10 min., then placing on ice. The reaction mix is purified using Performa DTR Gel Filtration Cartidges (Edge Biosystems, Inc.) as per manufacturer's protocols to remove excess linker. Linker ligation is verified by PCR amplification and gel visualization. Successful amplification is assumed if a "smear" of products is visible. The linker-ligated DNA is pre-amplified and PCI extracted, denatured, and hybridized with the repeat probes using the Dynabeads M-280 Streptavidin system (Dynal Co.) following manufacturer's protocols. The final eluted fragments are ethanol precipitated, and the entire pre-amplification and enrichment protocols repeated a second time. The enriched DNA is then pre-amplified and size fractionated using Sepharose CL-4B SizeSep 400 Spun Columns (Amersham Pharmacia Biotech, Inc.) as per manufacturer's protocols. Enriched fragments are cloned using the TOPO TA Cloning Kit (Invitrogen Corp.) as directed by the manufacturer. 96-well plates containing 100uls SOC broth w/100ug/ml Ampicillin are inoculated with transformed colonies and incubated overnight at 37C. 25 ul of cell culture from each well is transferred to a clean 96-well plate, pelleted via centrifugation, and resuspended in 50 ul of 10 mM Tris-HCl pH 8.0. The cloned fragments are amplified with M13 insert primers from the TOPO TA kit. After PCR amplification with the M13 forward & reverse primers, the products are visualized on an agarose gel to ensure that the amplification is successful. The products are then treated with exonuclease 1 prior to cycle sequencing. Cycle sequencing is accomplished with the T3 primer. 10 ul Hi-Di formamide is added to each well, and the plates incubated at 95C for 2 minutes, placed on ice immediately for about 10 min, then centifuged at 3500 rpm for 30 seconds prior to loading on an ABI 3100 Automatic Sequencer. The sequence files are analyzed using the GCG Blast protocol for the presence of repeat fragments & amplification primers are desinged from the flanking regions of the repeat. After successful fragment amplification.