Genes were cloned into the pCRII Topo vector using the TOPO TA cloning kit (dual promoter) according to manufacturer's instructions (Invitrogen), after amplifying genes of interest from a 24 hpf wild type embryo cDNA library.
Specific primers for the genes of interest amplifying average products of 300 bp with about 50 GC% and about 55°C Tm were designed (Additional file 3).
R.P.B. conducted all experimental procedures including mining microarray data and selecting genes of interest, amplifying, sequencing and analyzing the proximal promoters, conducted bioinformatics and statistical analysis, and drafted the manuscript.
Expression level changes were defined in 7 down-regulated genes and in 4 up-regulated genes by means of real-time PCR for fragments specifically amplifying transcripts of interest (Additional file 16), using GAPDH as a control for target gene expression normalization.
But our mission has to be about supporting and amplifying broad UK interests in our areas of work, and demonstrably providing benefit back to the UK, which I am confident we do.
We have a morbid fascination with catastrophe, so media companies have a vested interest in amplifying every horrific detail to make more money, unconcerned that this ultimately exacerbates our culture of fear and paranoia.
We created the latter by separately amplifying the regions of interest from each species then combining the amplicons into a 1∶1 mixture.
Each TaqMan genotyping assay contained two primers for amplifying the sequence of interest and two TaqMan® MGB probes for detecting alleles.
Horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) attracts growing interest as an amplifying label for biorecognition and biosensing events, especially for DNA detection.
The cDNA generated by this protocol was split into several reactions aimed at amplifying different outer products of interest.
Comparing both scenarios, one also finds that the overall magnitude of the competing CIF is reduced by amplifying the baseline CSH of interest.
