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A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for detection of - http://www.im.microbios.org/03setember98/08%20Sterflinger.pdf

Category: PCR Protocols: In-situ PCR Protocols

A protocol for application of Polymerase Chain Reaction (PCR) in situ. hybridization for the detection of hyphomycetes. Sterflinger et al 1998. - [Read A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for detection of]

ABI Prism 310 Cycle Sequencing Protocol - http://vosshall.rockefeller.edu/protocols/ABICYCLESEQ.pdf

Category: DNA Protocols: DNA Sequencing Protocols

Protocol for ABI prism 310 cycle sequencing. Includes: Sequencing Reaction; Column Purification; Sample Loading Preparation. - [Read ABI Prism 310 Cycle Sequencing Protocol]

AFLP genotyping and fingerprinting Review PDF - http://taxonomy.zoology.gla.ac.uk/~rdmp1c/teaching/L4/Evolution/Session5/Mueller.pdf

Category: PCR Protocols: AFLP PCR

Ulrich G. Mueller and L. LaReesa Wolfenbarger. Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for the rapid screening of genetic diversity. AFLP. TREE October 1999 - [Read AFLP genotyping and fingerprinting Review PDF]

Akt/PKB Kinase Assay - http://www.whitelabs.org/Lab%20Protocols/kinase%20and%20phosphatase%20assays/Akt%20PKB%20kinase%20assay.htm

Category: Signalling Signal Transduction Protocols

Protocol for Akt/PKB kinase assay. Protocol includes: Akt lysis buffer; Kinase Buffer; 1.5x Akt kinase reaction buffer; Start solution; Lipofectamine PLUS; CDNA synthesis (Roche #1483?88). - [Read Akt/PKB Kinase Assay]

Amino-allyl Labeling (GeneTAC) Protocol - http://www.bcm.edu/mcfweb/?PMID=3551

Category: Microarray Protocols: Amino-allyl Microarray Methods

Protocol for amino-allyl labeling (GeneTAC). Includes: RT Reaction; Nucleotide Mix for one reaction; Coupling; Prehybridization of probe. - [Read Amino-allyl Labeling (GeneTAC) Protocol]

Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4115

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR. A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems. The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Analyzing Yeast Colonies by PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4015

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Yeast colonies are suspended in complete PCR buffer and transferred to a thermal cycler for 35 cycles of PCR. The products of the amplification reaction are analyzed by gel electrophoresis. - [Read Analyzing Yeast Colonies by PCR Protocol]

Assay for Luciferase in Extracts of Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3642

Category: Reporter Gene Assays: Luciferase Assay Protocols

In this protocol, cells transfected with a luciferase reporter plasmid are lysed in a detergent-containing buffer. Luciferase in the extract catalyzes an oxidation reaction in which D-luciferin is converted to oxyluciferin, with production of light at 556 nm that can be quantified in a luminometer. - [Read Assay for Luciferase in Extracts of Mammalian Cells Protocol]

BAC-FISH Protocol - http://www.nhm.ac.uk/hosted_sites/schisto/protocols/BAC_FISH.html

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other manufacturers may work equally well but have not been tested. Includes: Labeling of BAC clones; Ethanol precipitation; Hybridization; Post-hybridisation treatment / detection. - [Read BAC-FISH Protocol]

Bidirectional Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4139

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for bidirectional, blunt-end cloning of DNA fragments. The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase. The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Bidirectional Cloning of PCR Products Protocol]

Blunt-end Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3830

Category: PCR Protocols: PCR Cloning Protocols

Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves. I - [Read Blunt-end Cloning of PCR Products Protocol]

Cell Viability Assays that Measure ATP Protocol - http://www.promega.com/paguide/chap4.htm#title2

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. The amount of ATP in cells correlates with cell viability. - [Read Cell Viability Assays that Measure ATP Protocol]

Chromatin Immunoprecipitation Protocol PDF - http://www.microarrays.ca/support/PDFs/ChIP_Protocol_for_Microarray_Analysis_Beads_09-07-04.pdf

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Chromatin Immunoprecipitation Protocol for Microarray Analysis â€“ Protein A/G Bead Method. Cross-Linking, Sonication/Chromatin Shearing Efficiency. Reaction UHN Microarray Centre - [Read Chromatin Immunoprecipitation Protocol PDF]

Colony PCR Protocol - http://www.enterococcus.ouhsc.edu/ColonyPCRProtocol.asp

Category: PCR Protocols: Colony PCR

PCR Reaction Conditions for Colony PCR. Contributed by Lynn Hancock. Enterococcus Research Site, The Board of Regents of the University of Oklahoma. - [Read Colony PCR Protocol]

Combined DNA In Situ Hybridization and Immunocytochemistry Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_127-134.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]

Competitive RT-PCR Protocol - http://www.uni-ulm.de/~dkaufman/rt-pcr.html

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for Competitive RT-PCR.For quantifying mRNA, we use a competitive RT-PCR protocol with internal standard RNAs. These are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is coamplified with the s - [Read Competitive RT-PCR Protocol]

Deglycosylation of Glycoproteins Using Endoglycosidases - http://www.glycotech.com/protocols/Proto3.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

Deglycosylation steps are different with every glycoprotein and must be determined empirically (Methods in Enzymology, 1994, 230: 44-57). A typical test reaction of human transferrin is described. T.H. Plummer, Jr. and A.L. Tarentino, Department of Biochemistry, New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany, New York - [Read Deglycosylation of Glycoproteins Using Endoglycosidases]

Detailed NF-kB EMSA Protocol - http://www.celldeath.de/apometh/emsa.html

Category: Cell Biology and Cell Culture: Apoptosis Protocols: NF-kB EMSA Protocols

Detailed NF-kB EMSA Protocol. Includes Nuclear extract preparation, Labeling of the oligonucleotide probe: T4 Polynucleotide Kinase Reaction, Supershift assay to identify NF-kB, and recipes for EMSA buffers. Celldeath.de - [Read Detailed NF-kB EMSA Protocol]

Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_216-219.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of even-skipped transcripts in drosophila embryos with PCR/DIG-labeled DNA probes. This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. Includes: Probe labeling; Evaluation of labeling reaction; Preparation of embryos, hybridization and detection. - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol]

Determination of Tissue Fungal Burden Utilizing Quantitative Real Time Polymerase Chain Reaction - http://www.sacmm.org/pdf/Determination%20of%20Tissue%20Fungal%20Burden%20utilizing%20Quantitative%20Real%20Time%20PCR.pdf

Category: Fungi Protocols: Fungi Genetics Protocols

Standard operating procedure for the determination of tissue fungal burden utilizing quantitative real time polymerase chain reaction (QPCR). This standard operating procedure will provide information on how to assess fungal tissue burden of infected animals by use of a single copy (FKS) or multicopy gene (18s RNA) to assess the number of fungal cell nuclei present. - [Read Determination of Tissue Fungal Burden Utilizing Quantitative Real Time Polymerase Chain Reaction]

Directional Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4140

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Directional Cloning of PCR Products Protocol]

Dye Protocols and Notes for Cosmid, BAC, BAC, Fosmid Templates - http://www.genome.ou.edu/big_dyes_cos_bac_pac_fos.html

Category: BACs Bacterial Artificial Chromosome Protocols

Sequencing conditions tested for the ABI Big-Dye terminators (PE-ABI #4303150 for the 1000 reaction kit - Description: TF,KIT BTD RR-1000) with various templates. Important to quantitate all templates by agarose gel electrophoresis vs size and concentration standards and do a few tests with different template concentrations to determine the optimal conditions for your reactions. Several conditions are given. - [Read Dye Protocols and Notes for Cosmid, BAC, BAC, Fosmid Templates]

ELISA Method to Measure Inhibition of the COX Enzymes Protocol - http://www.natureprotocols.com/2006/11/22/an_elisa_method_to_measure_inh.php

Category: ELISA Protocols

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. - [Read ELISA Method to Measure Inhibition of the COX Enzymes Protocol]

EMSA (Gel Shift) Technique Information - http://www.piercenet.com/Products/Browse.cfm?fldID=99A16E15-3470-4FD1-9A3C-0BBABBBE45F9

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

Information for EMSA (Gel Shift) technique. Includes: Critical EMSA Reaction Parameters. - [Read EMSA (Gel Shift) Technique Information]

EMSA Protocol using 32-p. - http://www.well.ox.ac.uk/flint/EMSA.htm

Category: DNA Protocols: EMSA DNA-Protein Protocols

Primer design, Primer annealing, Primer labelling, Probe purification, In vitro hybridization DNA-protein Binding reaction. Jonathan Flint. - [Read EMSA Protocol using 32-p.]

Articles (1-10 of 11)

Populational Analysis of Genomic DNA Protocol

A protocol on the populational Analysis of genomic DNA.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

DNA Ligation Protocol

The DNA Ligation protocol described here contains the steps required to join together using ligase enzyme both plasmid DNA and insert DNA fragments in order to create a new plasmid. This new ligated plasmid can be transformed after into competent bacteria to produce DNA for mini, midi or maxi-prep isolation.

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

Crystal Violet Stain

Crystal Violet stain preparation method and protocol.

Histone H1 Kinase Activity Assay

Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

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