PCR Quantitative real-time PCR

Get tips on using Direct-zol RNA Kits to perform RNA isolation / purification Cells - primary human renal proximal tubular epithelial cells

Isolating DNA from tissues and paraffin-embedded tissue samples can be challenging as double-stranded DNA is physically fragile and highly susceptible to exo- and endonucleases. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in the presence of DNAse inhibitors. Further, extracting DNA from the nucleus need specific methods by combining physical, mechanical and chemical lysis approaches,

Get tips on using PureYield™ Plasmid Miniprep System Technical Bulletin to perform Plasmid Isolation Agrobacterium tumefaciens

Get tips on using REPLI-g Mini Kit (100) to perform Whole Genome Amplification Parasites

Get tips on using pBYR2T-EGFP to perform Protein Expression Prokaryotic cells - A. tumefaciens GFP

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.

Get tips on using pSB 130 to perform Protein Expression Prokaryotic cells - A. tumefaciens ELP-intein

Get tips on using pJL TRBO-G to perform Protein Expression Prokaryotic cells - A. tumefaciens GFP

Get tips on using pBIN61:α-AIC3 to perform Protein Expression Prokaryotic cells - A. tumefaciens α-amylase inhibitor

Get tips on using pPZP-RCS2-ocs-nptII to perform Protein Expression Prokaryotic cells - A. tumefaciens α-amylase, amylopullulanase, and glucoamylase