Protein expression and purification Yeast Pichia pastoris

DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.

Get tips on using SurePrint G3 Human Gene Expression 8x60K v2 Microarray Kit to perform Microarray Human - PCOS

Get tips on using Unstained Protein Standards to perform Protein Ladder Unstained

Get tips on using Prestained Protein Standards to perform Protein Ladder Prestained

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 RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - Rat_Mesenteric fat

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - MLS-1765

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - BHK-21

Get tips on using pVRB2B3 to perform Protein Expression Eukaryotic cells - HEK293 hβ-defensin 2/3

Get tips on using pMAPLe3 to perform Protein Expression Prokaryotic cells - E. coli mycobacterial Esx complex