"The multiplex version is about six minutes shorter, not counting the intermission time, which is about 12 minutes".
The Django Unchained director clarified that he did not want to treat the multiplex version as a "left-handed version" so tweaked certain scenes to fit that setting.
Methylation-specific PCR (MSP) amplifies cancer-associated gene promotor methylation events, and the quantitative multiplex version of this assay (QM-MSP) has the capacity to detect 1 methylated copy among 100,000 unmethylated copies [ 50].
Using Multiplex Manager version 1.2 (Holleley and Geerts, 2009), initial test multiplexes were developed and new forward primers were synthesized.
The MSI Multiplex System Version 1.2 (Promega, Southampton, UK, cat. no MD1641) was used for the detection of microsatellite instability according to the instructions of the manufacturer.
Using the software Multiplex Manager version 1.2 (Holleley and Geerts, 2009), two 6-plex and one 5-plex multiplexes were designed, and new fluorescently labeled forward primers synthesized (Invitrogen; Table 2).
MSI analysis was performed using the MSI Analysis System (MSI Multiplex System Version 1.1, Promega) consisting of five nearly monomorphic mononucleotide markers (BAT-25, BAT-26, NR-21, NR-24, MONO-27) according to the manufacturer's instructions.
Libraries were prepared according to Agilent's protocol for SureSelect Target Enrichment for Illumina Paired-End Multiplexed Sequencing Version 1.0 (May 2010).
DNA was sheared to 300 bp using a Covaris S2 instrument and enriched by using the SureSelectXT Human All Exon 50 Mb kit and an Agilent NGS workstation according to the manufacturer's instructions (SureSelectXT Automated Target Enrichment for Illumina Paired-End Multiplexed Sequencing, version A, Agilent Technologies, Santa Clara, CA, USA).
Randomly fragmented DNA was end-repaired, extended with an 'A' nucleotide at the 3'end, ligated with the indexing-specific paired-end adapter and amplified according to the manufacturer's protocol (SureSelect Target Enrichment for Illumina Multiplexed Sequencing version 1.5; Agilent Technologies, Los Angeles, CA).
Running time for multiplex wide-release version: 2 47.
