Twitch says the feature was something that was designed based on requests from the community, and will continue to be iterated on throughout the year.
Primers were designed using reference sequence NT 030059 and will be provided on request.
All the coding exons plus the flanking intron-exon boundaries of CLU were PCR amplified using primers (available on request) designed using ExonPrimer software (http://ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html) and Roche FastStart PCR MasterMix polymerase (Roche Diagnostics Corp., IN).
Purified genomic DNA was aliquoted (10 ng/ul concentration) into 96 well plates and genotyped on a Sequenom™ system (Sequenom™ Autoflex Mass Spectrometer and Samsung 24 pin nanodispenser) by the Australian Genome Research Facility (www.agrf.com.au) using sequences designed in house (available on request) and recommended amplification and separation methods (iPLEX™, www.sequenom.com) [16].
For difficult to amplify templates, additional PCR primers were designed and are available on request.
Training vignettes were designed and are available on request from the website (http://sgdp.iop.kcl.ac.uk/opcritplus).ac.uk/opcritplus
Briefly, 20-bp primers flanking the given region and yielding amplicons of 300 600 bp were designed (primer sequences available on request).
The probes were co-designed by researchers at the Institute of Aquaculture, University of Stirling, U.K. and Nofima (Ås, Norway), and array design is available on request.
This resulted in a putative order of contigs surrounding the esp PAI, which was then confirmed by designing primers (sequences available on request) on the contig ends, followed by PCR (using AccuPrime™ Taq DNA Polymerase High Fidelity; Invitrogen, Breda, The Netherlands) and Sanger-sequencing of the generated PCR products.
The engineered miRNA directed against the mRNA of NG2 (the sequence is available on request) was designed using the BLOCK-iT RNAi Designer software of Invitrogen and cloned following the BLOCK-iT™ Pol II miR RNAi Expression Vector kit (Invitrogen) into the gateway cassette (Invitrogen) of the pCDB-GW vector together with the EmGFP cDNA.
