content Protocols

(LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages.

3'-End cDNA Amplification Using Classic RACE Protocol To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO).  Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1).

5'-End cDNA Amplification Using Classic RACE Protocol To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT).  Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP.  Amplification is carried out using three primers.

5'-End cDNA Amplification Using New RACE Protocol New RACE, a variation of RNA ligase-mediated-RACE (RLM-RACE) (Liu and Gorovsky 1993) departs from classic RACE (see 5'-End cDNA Amplification Using Classic RACE) in that an "anchor" primer is attached to the 5'-end of the mRNA before the reverse transcription step; hence the anchor sequence becomes incorporated into the first-strand cDNA if, and only if, the reverse transcription proceeds through the entire length of the mRNA of interest.

6-Azauracil Sensitivity Assay for Yeast Protocol Protocol describes an assay where it requires growing saturated cultures of yeast, counting, and spotting serial dilutions of yeast on both CSM and CSM + 6AU plates.

A Batch Test Tube Method for the Calculation of an Adsorbent’s Available Capacity Protocol Protocol describes a batch test tube method for the calculation of an adsorbent’s available capacity.

A Magnetic Particle-Based Method for Purifying PCR Products from Solution Protocol The MagneSil system can selectively isolate PCR products that are more than 150-bp long from primers and primer -dimers.  The technology can be used with a number of robotic workstations, including Beckman Coulter’s Biomek 2000 and FX Laboratory Automation Workstations.  The procedure can also be carried out manually.  Typical recovery is more than 80% for a 1-kb product with negligible carryover of primers or nucleotides.

A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor.

A Novel Fluorescent pH Probe for Expression in Plants The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.

A Novel Method for Simultaneous Anterograde and Retrograde Labeling of Spinal Cord Motor Tracts Protocol for a method for simultaneous anterograde and retrograde labeling of spinal cord motor tracts in the same animal.

A Sealed Preparation for Long-Term Observations of Cultured Cells This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO2 control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours.

A Sealed Preparation for Long-Term Observations of Cultured Cells: Support Slide Construction Protocol on the details required of support slide construction for a sealed preparation for long-term observations of cultured cells.

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements Yang et al, NAR. 2004. ue to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deox

A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O.

Actin Staining in Fixed Yeast Cells Protocol Phalloidin binds specifically to F-actin, and fluorescent-tagged phalloidin stains the actin skeleton in cells in a manner that is very close to the staining pattern seen using anti-actin antibody.

Advances in Cytochemical Methods for Detection of Apoptosis Advances in Cytochemical Methods for Detection of Apoptosis. Katherine L. Barrett et al. 2001

Affinity Purification of Interacting Proteins from Cell Lysates Protocol Recombinant protein or a chemically synthesized bioactive fragment is immobilized on resin and used as a probe to capture interacting proteins directly from a cell extract.  Affinity-purified proteins are fractionated by gel electrophoresis and visualized by Coomassie staining.  Proteins that interact specifically are identified by comparing this gel profile to one obtained from cell lysates passed over a control resin lacking the immobilized probe protein.

Agarose Gel Electrophoresis Protocol Protocol on how to pour, load, and run an agarose gel.

Alkaline Agarose Gel Electrophoresis Protocol Alkaline agarose gels are used chiefly to measure the size of first and second strands of cDNA (Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase) and to analyze the size of the DNA strand after digestion of DNA-RNA hybrids with nucleases such as S1.

Alkaline Phosphatase Treatment of Bacteriophage lambda Vector DNA Protocol Removal of the 5'-phosphate groups from the internal termini of bacteriophage {lambda} arms is used to prevent self-ligation and suppress the background of nonrecombinant bacteriophages during cloning.

Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture Protocol Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. If amplification should become necessary, it is best to expand the library by growth in liquid medium.

Amplification and Storage of a Cosmid Library: Amplification on Filters Protocol Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. In this method of amplification, distortion of the library is rarely a problem because at no stage are bacteria containing different recombinant cosmids grown in competition with one another.

Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs

Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium 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.

An Assay for RNA-Dependent RNA Polymerase Activity Protocol Protocol provides a quick way to assay for RNA-dependent RNA polymerase (RdRP) activity in crude protein samples.  RdRP transcription products remain at the origin during paper chromatography.