A new method of extracting bacterial and yeast DNA from blood products dependent on guanidinium thiocyanate acid extraction and proteinase K treatment is described. In spiked samples the sensitivity per 0.1 ml of serum and blood, respectively, was 26 and 150 colony forming units (cfu) for Escherichia coli, 80 and 120 cfu for Staphylococcus aureus and 20 and 26 cfu for Candida albicans. This com...

The analysis of genomic DNA is widely-used for research, forensic and diagnostic purposes. Here we describe reliable methods for isolation of cell-free DNA and cellular DNA from urine. Both DNA fractions are suitable for PCR and Methylation-Specific PCR (MSP) amplification, leading to consistent and reproducible results. A kinetics analysis describes the decline of efficiency of MSP performed w...

proteinase K and was then incubated for 4 hrs at 55•Ž . After approximately 30 mg of nail clippings in a 1.5 ml microtest tube were rinsed once with 1.0 N NaOH and twice with distilled water, the nails were mixed with 300ƒÊ1 of 4 M guanidine thiocyanate, 0.5% (w/v) sodium N-lauroyl salcosinate, 25 mM sodium citrate, and 0.1 mM 2-mercaptoethanol [7], and were incubated at room temperature over n...

The Streptococcus cremoris Wg2 proteinase gene, cloned in S. lactis, specified a proteinase which exhibited the same specificity toward casein as did the proteinase isolated from the original host. Although the cloned gene lacked the last 130 codons, the proteinase still specifically degraded beta-casein. Deletion of the C-terminal 343 amino acids from the proteinase did not influence this spec...

A supernatant fraction was prepared from rat uterine myometrium by homogenization, sonication and centrifugation. In this supernatant the protein concentration and the activities of an acid proteinase, an acid phosphatase and a proteinase inhibitor were measured. From the fibrous sediment, after washing with 0.5% Triton X-100 and with water, an actomyosin-containing solution was obtained by ext...

sue samples using Proteinase K digestion method. A 5 ug of the viral RNA was used for the synthesis of cDNA using RevertAid TM First Strand cDNA Synthesis Kit (MBI, Fermentas). The VP2 hypervariable region of the IBDV was amplified using specific primers. A single band of PCR product of the expected size was visualized on 1.5% agarose gel after ethidium bromide staining. Results indicated that ...

It is demonstrated that a diagnostic PCR for Borrelia burgdorferi can be inhibited in the presence of more than 500 ng of host (monkey skin) DNA. The inhibitor is the host DNA itself. An acceptable value for analytical sensitivity can be obtained by diluting the skin-B. burgdorferi proteinase K lysate to a level below the inhibitory concentration of the host DNA. Dilution of the lysate may obvi...

We have identified and characterized three stage-specific proteinases of Plasmodium falciparum that are active at neutral pH. We analyzed ring-, trophozoite-, schizont-, and merozoite-stage parasites by gelatin substrate PAGE and characterized the identified proteinases with class-specific proteinase inhibitors. No proteinase activity was detected with rings. Trophozoites had a 28 kD proteinase...

Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. A...

In this study the extracellular proteins from isolate LC3 belonging to Aspergillus were purified for new antimicrobial polypeptide (AMP) discovery and then tested activity against Staphylococcus aureus (ATCC 25923) Methicillin-resistant S. (MRSA). Antimicrobial was determined by trypsin/proteinase K assay, which polypeptide-based, it observed that protein a of about 11 kDa gel overlay assay. Th...