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Preparation of Plasmid DNA by Lysis with SDS Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3906

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS. - [Read Preparation of Plasmid DNA by Lysis with SDS Protocol]

Preparation of Plasmid DNA: Toothpick Minipreparation Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3905

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. - [Read Preparation of Plasmid DNA: Toothpick Minipreparation Protocol]

Preparation of Single-stranded Bacteriophage M13 DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3686

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. - [Read Preparation of Single-stranded Bacteriophage M13 DNA Protocol]

Preparing Cell Smears from Tissue Samples for Immunostaining Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4330

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Staining of tissues can be carried out on cell smears prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. - [Read Preparing Cell Smears from Tissue Samples for Immunostaining Protocol]

Preparing Paraffin Tissue Sections for Immunostaining Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4329

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Most histological studies are carried out on paraformaldehyde-fixed, paraffin-embedded tissue samples. Therefore, there is an extensive atlas of most tissues and organs prepared from these sources, and comparing the location of antigens to these data is immediately informative. The fixation and embedding procedures are harsh, however, and many antigens are not well preserved. - [Read Preparing Paraffin Tissue Sections for Immunostaining Protocol]

Preparing Stocks of Bacteriophage {lambda} by Small-scale Liquid Culture Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3971

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

High-titer stocks of bacteriophage {lambda} are easily prepared by infecting small-scale bacterial cultures. - [Read Preparing Stocks of Bacteriophage {lambda} by Small-scale Liquid Culture Protocol]

Protocol Spindle Assembly In Vitro - http://www.bio.com/protocolstools/protocol.jhtml?id=p1051

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes an in vitro reaction to assay mitotic spindle assembly. The assay uses Cytostatic Factor extract made from Xenopus eggs, fluorescently-labelled tubulin, and prepared sperm nuclei. Spindle assembly is monitored by immunofluorescence microscopy. - [Read Protocol Spindle Assembly In Vitro]

Purification of BAC DNA Prepared with the NucleoBond System Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4391

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol describes purification of BAC DNA that has been prepared with the commercial NucleoBond system. - [Read Purification of BAC DNA Prepared with the NucleoBond System Protocol]

Rapid Isolation of Mammalian DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3514

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Mammalian DNA prepared from blood or tissues as described in this protocol is 20-50 kb in size and suitable for use as a template in PCRs. The yields of DNA vary between 0.5 and 3.0 µg/mg tissue or 5 and 15 µg per 300 µl of whole blood. - [Read Rapid Isolation of Mammalian DNA Protocol]

reparation of Copy Standards for Southern Blot Copy Number Determination - http://www.med.umich.edu/tamc/spike.html

Category: Transgenic Mouse Protocols: Mouse Genotyping Protocols

PCR screens must be designed to detect transgene DNA at the single copy level.Copy standards are prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA to produce transgene copy standards. University of Michigan Transgenic Animal - [Read reparation of Copy Standards for Southern Blot Copy Number Determination]

S20 Purification of detergent-insoluble lipid rafts from cells and tissues. - http://www.axis-shield.com/densityhome/optiprep/S20.pdf

Category: Cell Biology and Cell Culture

Protocol for the isolation of the lipid-rich microdomains of the plasma membrane, notably caveolae and lipid rafts. Methods for the isolation of lipid rafts are based on the insolubility of these structures in the nonionic detergent TritonX-100. Either the intact cells are treated with a detergent-containing solution or a post-nuclear supernatant is prepared from a cell homogenate and then Triton X-100 is added to this supernatant. - [Read S20 Purification of detergent-insoluble lipid rafts from cells and tissues.]

Tissue sectioning and in situ hybridization as prepared by Simon Dunn PDF - http://oregonstate.edu/~weisv/Protocols/Cellular/Tissue%20sectioning%20and%20in%20situ%20hybridization%20Protocol.pdf

Category: Histology Protocols: Tissue Sample Sectioning

Tissue sectioning and in situ hybridization as prepared by Simon Dunn PDF. Includes Reagents and Solution recipes. Weis Lab, Virginia. - [Read Tissue sectioning and in situ hybridization as prepared by Simon Dunn PDF]

Transformation of E. Coli by Electroporation Protocol. - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3933

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Electrocompetent bacteria are prepared by growing cultures tomid-log phase, washing the bacteria extensively at low temperature,and then resuspending them in a solution of low ionic strengthcontaining glycerol. DNA is introduced during exposure of thebacteria to a short high-voltage electrical discharge.

- [Read Transformation of E. Coli by Electroporation Protocol.]

Yeast Plasma Membrane H+ -ATPASE Toxcity Test Protocol - http://embryo.ib.amwaw.edu.pl/invittox/prot/34.htm

Category: Yeast Protocols

Plasma membranes are isolated from the yeast Saccharomyces cerevisiae. The cell wall is initially digested by helicase, followed by hypoosmotic lysis and homogenization. Membranes are prepared by subsequent differential centrifugation. The activity of the H+-ATPase is then determined by measuring the amount of inorganic phosphate released from ATP. - [Read Yeast Plasma Membrane H+ -ATPASE Toxcity Test Protocol]

Articles (1-8 of 8)

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.

Microinjection Pipette Pulling Protocol

A simple protocol for microinjection pipette pulling.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

Coupling Cysteine Containing Peptides to Carrier Protein for Immunization and Polyclonal Antibody

This protocol is used in the production of polyclonal antibodies that recognize a peptide sequence of interest.

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.

Picking Transfected ES Cell Clones Protocol

This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.

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