180x Filetype PPT File size 0.94 MB Source: uvm.edu
The following hybridization mix is prepared for each sample Fragmented cRNA 5ug 10 ul Control B2 Oligo 1.7 ul 20x Eukaryotic Control mix [bio B, bio C, bio D, Cre] 5 ul Herring Sperm DNA [10mg/ml] 1 ul Acetyleted BSA [50mg/ml] 1 ul DMSO 10 ul 2x Hybridization Buffer 50 ul Water 22.3 ul Denature 99C Inject into 10 minutes GeneChip The hybridization oven Target binds to the Probes RNA-DNA Hybridization Targets: Antisense biotinylated cRNA Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond Hybridization Optimized Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequence [We hope] Types: DNA to DNA DNA to RNA RNA to RNA PNA to DNA Stringency Stringency is a condition that causes a change in the local hybridization environment and “interferes” with the binding kinetics Stringency prevents: . Binding of non-complementary strands Self hybridization – hairpin formation Disassociation of strands
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