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ORF-specific primer design and strategic 96-well plate organization ORF sequences were imported into the FLEXGene LIMS and primer sequences were designed using a nearest neighbor algorithm. Primer lengths were adjusted to adhere to a common melting temperature of 58°C. Moreover, start codon sequences were normalized to ATG (Met) and natural stop codons were replaced with TTG (Leu) in the final primer designs. In addition to ORF-specific, start and stop regions, the 5’ and 3’ primers included fixed sequences that correspond to partial att sequence-specific recombination recognition sites that flank the ORF in the resultant plasmid clones. To organize the positions of individual ORFs on production plates prior to PCR amplification, each ORF was first grouped in the database based on its size in the FLEXGene LIMS, as PCR amplicons of different sizes were treated differently in the clone production pipeline (see below). The first group comprises all ORFs larger than 2,000 bp and the second group, ORFs 2,000 bp or smaller. Additionally, the ORFs were arranged on 96-well production plates in a saw-tooth pattern. That is, ORFs were ordered by predicted PCR amplicon size and then arranged in a pattern that alternates larger and smaller amplicon sizes. This was useful both, for visual distinction of successful PCR amplicons after agarose gel analysis, and to reduce the chance of cross-contamination when cloning steps include gel purification. Oligonucleotide primers were purchased from Integrated DNA Technologies as 50M normalized solution and diluted prior to use in PCR reactions to 0.083M. PCR and capture in the entry vector pDONR221 PCR was performed in barcode-labeled, 96-well plates and verified by agarose gel electrophoresis. For ORFs >2,000 bp the PCR amplicons were also isolated from the gel and purified prior to capture in linearized entry vector. For ORFs greater than 2,000bp, we used the In-Fusion strand displacement method (Clontech) after a single round of PCR, for which we developed a variant of pDONR221 that allows for linearization with two restriction endonucleases adjacent to the 5’- and 3’-att-site sequences. This methode of capture requires the removal of any free dNTPs, hence we purified the amplicon by excision from the agarose gel and spin-filtration over 25M melt-blown polypropylene 96-well filter plates (Whatman). For ORFs less than or equal to 2,000 bp, we used the BP Clonase-based capture method (Invitrogen). Because this method requires an intact, full-length att–site sequence, a second round of PCR was performed using oligonucleotide primers (Forward: 5’-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC C; Reverse: 5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC) that recognize the partial att-site sequence common to all first-round PCR amplicons and generating optimized BP-capture amplicons. Clonal isolation and production of glycerol stocks Independent of capture method, transformations into E. coli were plated to 48- sector LB/agar dishes with the appropriate antibiotic selection and grown overnight at 37°C. Colonies were visualized and counted, and single isolates from each sector were robotically picked for inoculation into 1mL growth media (LB/antibiotic) using a customized Megapix robot (Genetix), and cultures were grown overnight at 37°C. Inoculated cultures were assayed for growth via OD600 measurement and aliquots were stored as 15% glycerol stocks. Plasmid DNA preparation, sequencing and analysis Plasmid DNA was prepared using a 96-well silica preparation method as described elsewhere (references?). DNA sequencing was performed at the Dana Farber/Harvard Cancer Center DNA Resource Core (http://dnaseq.med.harvard.edu) using standard M13-forward and -reverse primer, or ORF-specific primers where necessary (sequences available upon request). Sequence data and ORF reference sequences were imported into the Automated Clone Evaluation (ACE) suite of software tools (http://bighead.harvard.edu:8080/ACE; Taycher et al., in preparation). ACE was then used to perform all DNA sequence and clone evaluation steps, including sequence quality assessment, sequence contig assembly, identification of sequence coverage gaps, design of oligonucleotide primers for internal sequencing, conceptual translation of experimentally determined sequence contigs, sequence comparison, and automated acceptance and rejection of individual clone samples based on user-defined rules for minimal and maximally acceptable differences between the experimental and reference sequences at the nucleotide and amino acid levels.
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