PCR Cloning: shRNA-encoding vector construction

Altogen Labs offer cloning services, DNA sequencing, alignment, and plasmid construction. Customers are welcome to send Altogen Labs the Gene ID/Genbank/Swiss-Prot number, the gene/protein sequence, or a plasmid containing a gene of interest.

shRNA Design and Synthesis

Altogen Labs offers a complete service for preparing DNA templates and synthesizing hairpin shRNAs. Purification of the resulting shRNAs and preparation for transfection into cultured cells are offered as well.

mRNA Amplification

Altogen Labs uses the T7 promoter (tagged polydT) primer for priming first strand cDNA synthesis. Double-stranded cDNA is used as a template for in vitro transcription. In addition to standard ribonucleotides, company scientists also incorporate modified nucleotides (e.g., aminoallyl-UTP) to produce labeled RNA probes and amplifies the original mRNA more than 1 million-fold. RNA is also purified and prepared for all additional applications.

Gene Knockdown by shRNAs

The customer provides the name and accession numbers of the target genes, mails the cells and primary antibodies for Western blotting analysis to Altogen Labs, and the lab team does the rest . Altogen’s knockdown service includes design, construction, and confirmation of the 3'-5' sequence the shRNA vectors for every target gene. Design and synthesis of the SYBR-labeled qPCR primers for quantitation of target gene expression, shRNA transfection into the cell line supplied by the customer, and targeted gene expression analysis by qPCR at 24, 48, and 72 hr post-transfection and Western blot at 48 and 72 hr after transfection are performed by company scientists. Altogen Labs guarantees 70% or more inhibition of target gene expression. Transfection controls includes the negative vector control with scrambled sequences (21 bp) and a GFP protein vector control for assessment of transfection efficiency.