Journal: Journal of Lipid Research

Article Title: Splice donor site mutations in the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene cause a deficiency of the endoplasmic reticulum 3-hydroxy-3-methylglutaryl coenzyme A reductase protein in UT2 cells

doi: 10.1016/s0022-2275(20)32473-1

Figure Lengend Snippet: Fig. 9. Genomic Southern blot analysis of CHO, UT2 and UT2* cells. Genomic DNA from CHO, UT2, and UT2* cells was digested with XbaI (lane 1), BamHI (lane 2), and EcoRI (lane 3) and the digests (12 ug of DNA/lane) were electrophoresed in a 0.9% agarose gel for 5 h at 85 V. The DNA was then transferred to Bio-Rad’s Zeta-Probe blotting membrane and hybridized with four different cDNA inserts corre- sponding to exons 2, 11, 12, and 39 UTR of hamster HMG-CoA reductase. Probe sequences were verified by sequencing. Probes were nick- translated with 32PadCTP and hybridized overnight at 428C. Hybridization buffer consisted of 50% formamide, 7% SDS, 0.12 m Na2HPO4 and 0.25 m NaCl. Washings for all probes were at 558C with 0.5 3 SSC solution. For exon 12 washing was also done at 728C (high stringency). Blots were exposed with a Molecular Dynamics Phosphorimager.

Article Snippet: Electrophoresis supplies, AG1-X8-200-400 mesh formate resin, Zeta Probe GT membrane (used for Northern analysis) and Trans-Blot Transfer Medium (used for Western analysis) were purchased from BioRad, except for Protogel, which was purchased from National Diagnostics (Atlanta, GA).

Techniques: Southern Blot, Agarose Gel Electrophoresis, Membrane, Sequencing, Hybridization

Journal: Molecular Microbiology

Article Title: Mycoplasma pneumoniae Community Acquired Respiratory Distress Syndrome toxin expression reveals growth phase and infection-dependent regulation

doi: 10.1111/j.1365-2958.2010.07092.x

Figure Lengend Snippet: Transcription of cards during in vitro growth of M. pneumoniae in SP-4 broth. A. Expression of cards along with other genes was analysed by DNA slot blot. M. pneumoniae gene-specific PCR products were blotted onto Zeta probe membranes. M. pneumoniae S1 cells were grown and harvested at early-log (24 h), mid-log (48 h), late-log (72 h) and stationary phases (120 h). 32 P-labelled cDNAs generated by reverse transcription of isolated total RNA were used as hybridization probes. Experiments were repeated three times. B. Expression of cards by relative real-time quantitative RT-PCR (qRT-PCR). M. pneumoniae S1 cells were grown and RNA isolated at specific growth phases. Real-time qRT-PCR was performed using SYBR green chemistry as detailed in Experimental procedures . Experiments were repeated two times. The average fold differences in expression levels of cards mRNA at various time points (when compared with the stationary phase) and standard deviations (SD) are presented.

Article Snippet: All generated amplicons were purified by gel extraction, and 200 ng of each specific PCR product was blotted in triplicate on Zeta probe membranes by using Bio-Dot SF microfiltration apparatus as suggested by the manufacturer (Bio-Rad Laboratories, Hercules, CA).

Techniques: In Vitro, Expressing, Dot Blot, Generated, Reverse Transcription, Isolation, Hybridization, Quantitative RT-PCR, SYBR Green Assay