Monday, November 7, 2011
AM
-Welcome and tour of the AVRL laboratory facilities
-Introduction to MHC genotyping in nonhuman primates
-Isolation of total cellular RNA
-Overview of Roche/454 pyrosequencing
PM
-Preparation of SuperScript III 1st strand cDNA
-Generation of primary cDNA/PCR amplicons
-Verification of amplicons by agarose gel electrophoresis
Tuesday
AM
-Purification of primary amplicons with AMPure XP beads
-Quantitation of primary amplicons with Pico Green (Qubit)
PM
-Normalization and pooling of primary amplicons
-Quality control of amplicon pools - Agilent Bioanalyzer
-Preparation of emulsion PCR reactions (up to 3 pools in parallel)
Wednesday
AM
-Purification of emulsion PCR beads (break & enrich up to 3 libraries in parallel)
PM
-Information management – upload library sample info to LabKey database
-Preparation and loading of picotiter plate #1
-Initiation of Roche/454 GS Junior run #1
Thursday
AM
-Examination GS Junior run #1 results, MID binning and quality metrics
-Introduction to approaches for 454 genotyping (BLAST, BLAT, Galaxy pipeline)
PM
-Introduction to novel allele discovery (Geneious Pro 5.5 software)
-Preparation and loading of picotiter plate #2
-Initiation of Roche/454 GS Junior run #2
Friday, November 11, 2011
AM
-Examination GS Junior run #2 results
-Application of project specific software for sequence analysis of initial runs
PM
-Preparation and loading of picotiter plate #3
-Initiation of Roche/454 GS Junior run #3
-Extension of sequence analysis with project specific software
-Departure for airport as necessary
