Cell Based Reagents
 

CHI Scientific Inc. is offering high quality total RNA, PCR-ready first strand cDNA, proteins subcellular components derived from fine prepared mouse, rat and normal and diseased matchpair™ human primary cells. These high quality primary cell derivatives provide an excellent tool for validating data obtained from cell line-based experiments as well as various biomedical research experiments.

Total RNA Preparation: Total RNA used for cDNA synthesis is isolated by Trizol, Qiagen RNeasy kit followed by Qiagen RNA Clean Kit.
cDNA Preparation: The first-strand cDNA is synthesized from total RNA isolated from variety of animal and human primary cells at all ages. Ten micrograms of total RNA is primed with random primer oligonucleotides and reverse-transcribed by MMLV reverse transcriptase in 50 ul final volume. RT reaction is stopped by heating at 68 C for 10 minutes. The cDNA is delivered in 1x RT buffer. (1x RT Buffer: 50 mM Tris-HCl, pH 8.3, 75 mM KCI, 3 mM MgCI2, 10 mM DTT). 1 ul cDNA is sufficient for one PCR reaction. The 5' end of animal or human beta-actin cDNA has been amplified as QA/QC analysis by PCR from all cDNAs prior order shipment.

Protein Preparation: Fresh primary cell lysate was prepared in modified RIPA buffer (50mMTris, pH7.2, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% NP-40 , 1mM Na3VO4, 5mM NaF, 400uM PMSF, 1ug/ml of pepstatin A, 5ug/ml of aprotinin, 5ug/ml of leupeptin). Tissue debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The tissue protein was diluted and boiled for 5 min with non-reducing protein buffer (50 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS, 0.002% bromphenol blue) containing 5% beta-mercaptoethanol.

Applications: (1) PCR amplification of known genes; (2) Analysis of mRNA alternative splicing; (3) Gene cloning and target sequencing; (4) Western and Northern Blot; (5) Protein Arrays and Proteomics; (6) Chip Study and Expression profiling.