ProCipitate™ Applications

Human Fibroblasts

Stephanie M Cohen, Terrence S Furey, Norman A Doggett, and David G Kaufman. Genome-wide sequence and functional analysis of early replicating DNA in normal human fibroblasts BMC Genomics.2006;7: 301

Procipitate™ was used in a study that replicated mammalian genomic DNA during the S phase. Scientists created a cosmid library containing DNA enriched in sequences to replicate early in the S phase of normal human fibroblasts. Next the sequencing and alignment of the clone ends to the human genome occurred. Cosmid DNA template was purified in 96-well format using ProPrep™ and ProCipitate™ from Biotech Support Group.

Plasmid DNA

G.M. Huang; K. Wang; C.L. Kuo; B. Paeper; L. Hood A High-Throughput Plasmid DNA Preparation Method Analytical Biochemtry.1994;15;223(1):35-8

Researchers used ProPrep™ BAC 96 for plasmid DNA preparation based on alkaline denaturation in a 96-well format. Purification is done by ProCipitate™ in 96-well micro filters. Using this method scientists optimized the right bacterial strain for plasmid yield and did further analysis of different isoforms. The yield and quality of DNA are sufficient for restriction digestion, radioactive sequencing, and automated fluorescent sequencing to ultimately develop new medicines for gene therapy. The steps involve an alkaline lysis miniprep to screen bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. Next producing milligrams of plasmid DNA and purifying the crude lysate by centrifugation. 96 samples can be purified from crude tissue digests and are produced in microtitre plate format to allow efficient downstream processing of samples.

Bacterial artificial chromosome (BAC) vector

J M Kelley, C E Field, M B Craven, D Bocskai, U J Kim, S D Rounsley, and M D Adams. High throughput direct end sequencing of BAC clones.Nucleic Acids Res. 1999 March 15; 27(6): 1539–1546.

Bacterial artificial chromosome (BAC) vector generated libraries are popular for forming clone sets in high throughput genomic sequencing projects primarily because of their high stability. A low cost, efficient, 96 well procedure for BAC end sequencing for creating BAC end sequences from human and Arabidoposis libraries with high accuracy is described in this article. To develop a basic alkaline lysis filter plate that is processed in a 96 well template purification procedure yielding BAC DNA for several sequencing reactions and a DNA fingerprint, researchers used Procipitate™ from Biotech Support Group. Procipitate™ bound to proteins, removed it and helps with proper flow through the filter plates.

Recombinant Bacterial Colonies

Reddy, O.U., Pepper, A.E., Abdurakmonov, I., Saha, S., Jenkins, J.N., Brooks, T., Bolek, Y., El-Zik, K.M. New Dinucleotide And Trinucleotide Microsatellite Resources For Cotton Genome Research. Journal Of Cotton Science. 2000. V.5.P.103-113

Researchers used Procipitate™ to streamline the process of microsatellite capture and characterization for the development of molecular markers. After isolating recombinant bacterial colonies in a 96-well 0.6-mL-deep plate, cultures were put into high-density shaking-incubator and the bacteria pelleted, centrifuged and resuspended in a plasmid mini-prep solution containing RNaseA . Next the cells were lysed and Procipitate™ reagent was added to precipitate proteins and chromosomal DNA. The supernatent was filtered, precipitated, and sequenced.

DNA Extraction Genetic Diversity as an Indicator of Ecosystem Condition and Sustainability for Regional Assessments of Stream Condition in the Eastern United States

For DNA purification, a portion of the frozen tail section (approximately 25 mg) was homogenized in a 1.5 ml microfuge tube containing 100µl PBSET (standard PBS, 100mM EDTA, 0.1% Triton X). 400µl of PBSET, 27µl Proteinase K (20mg ml -1 ) and 30µl of 20% SDS was added to the homogenate. The tube was mixed by gentle inversion and incubated overnight at 65 °C. Following digestion, the preparation was centrifuged at 12,000 rpm for 10 minutes to remove particulate and undigested material. The supernatant was removed to a clean microfuge tube, incubated for 10 minutes at 91 °C to inactivate the Proteinase K, then allowed to cool to room temperature. 10 µl of RNAse(A) (20mg ml -1) was added to the supernatant and incubated for 30 minutes at 42 °C. 1 ml of ProCipitate™ was added to the tube, which was then mixed by gentle inversion on a rotator for 5 minutes and centrifuged at 14,000 rpm for 15 minutes at room temperature. To facilitate the separation of the DNA-containing supernatant and the semi-solid, protein-containing pellet, a dab of light PhaseLock gel (5'-3', Inc.) was added to the inside of the microfuge cap and the sample was centrifuged for an additional 15 minutes at 14,000 rpm at room temperature.

Quiniou SM; Katagiri T; Miller NW; Wilson M; Wolters WR; Waldbieser GC Construction and characterization of a BAC library from a gynogenetic channel catfish Ictalurus punctatus. Genetics, selection, evolution : GSE. 2003;35(6):673-83

Bacterial artificial chromosome (BAC) libraries are used as entire genomes for large DNA insert clones because they are stability, require low levels of chimeric inserts, and ease of use. A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Ten CCBL1 clones that did not contain an insert after restriction digestion. analysis were grown overnight in 5 mL LB/chloramphenicol. The BAC DNA was prepared using 100 mL Procipitate™ from Biotech Support Group to the neutralization buffer during the alkaline lysis protocol and all steps were performed at RT. Precipitated DNA was air-dried and resuspended in 30 mL of water.

Mycobacterium Avium Paratuberculosis

Charles G. Thornton; Kerry M. MacLellan; Judith R. Stabel; Christine Carothers; Robert H. Whitlock; Selvin Passen. Application of the C18-Carboxypropylbetaine Specimen Processing Method to Recovery of Mycobacterium avium subsp. paratuberculosis from Ruminant Tissue Specimens. Journal of Clinical Microbiology.2002;40(5):1783-1790

Scientists used Procipitate™ in specimens for a method for processing human respiratory specimens using the Zwitterionic detergent C18-carboxypropylbetaine (CB-18) was reported to improve the recovery of mycobacteria. Paraffin-embedded sections for each tissue are placed into RNase- and DNase-free microcentrifuge tubes. Pellets resuspended in buffer, EDTA and Tween. Proteinase K (0.2 mg/ml) was added to each tube, and samples were incubated overnight at 42°C. to inactivate the proteinase K. After centrifugation, an equal volume of ProCipitate™ is added to the supernatant of the sample being extracted, and samples were mixed gently for 5 min, left for 1 min, and centrifuged for 2 min at 16,000 g. Twenty-five microliters of 4 M LiCl was added to the supernatant, followed by 750 l of cold 100% ethanol. Samples were mixed by inversion and placed in a 70°C freezer for 30 min to 1 h. DNA pellets were resuspended in 200 l of Ultrapure RNase- and DNase-free H2O were amplified using the PCR amplification.

Enteric Viruses

Schwab KJ; De Leon R; Sobsey MD. Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR. Applied and Environmental Microbiology.1996;62(6):2086-94

To assess the risks from viral contamination of drinking-water supplies scientists used Procipitate™ for 1 liter of 1% beef extract-0.05 M glycine (BE/G) eluate that was concentrated and purified by polyethylene glycol (PEG) precipitation for the developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection.

Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR. Applied and environmental microbiology.1995;61(2):531-7

Researchers used Procipitate for a concentration and purification procedure to facilitate reverse transcription (RT)-PCR detection of enteric viruses in water of beef extract-glycine eluate with or without humic acid and seeded with poliovirus type 1, hepatitis A virus, and Norwalk virus. The method involved using polyethylene glycol precipitation, ProCipitate™ precipitation, a second polyethylene glycol precipitation, spin column chromatography, and ultrafiltration. Virus adsorption to and elution from ProCipitate™ compared with as a virus purification step against PEG-precipitated samples by combining equal volumes of ProCipitate™, mixing the preparations for 15 min, and centrifuging them. The use of a commercially available protein-adsorptive reagent, ProCipitate™, for purification of PEG precipitates resulted in effective virus recovery and sample purification and dramatically improved virus detection by the RT-PCR. The overall procedure included PEG precipitation, ProCipitate™ precipitation, a second PEG precipitation, spin column chromatography, and ultrafiltration.

Detection of Mycobacterium avium subspecies avium in formalin-fixed, paraffin-embedded tissues of captive exotic birds using polymerase chain reaction

Proprep™ for Large Scale Extraction and Purification of Clinical-Grade Plasmid DNA for DNA Vaccines from Biotech Support Group

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Using Proprep ™ For High-Throughput BAC & Plasmid DNA Isolation and Bioprocessing from Biotech Support Group