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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]

B- Galactosidase Assay Protocol - http://www.biochem.northwestern.edu/ibis/morimoto/research/Protocols/VI.%20Transcription/I.%20b-gal%20.pdf

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The recommended amount of RSV-ß-Galactosidase plasmid to use for transfection of cells (60 mm or 100 mm dish) is 1-2 µg. The optimal amount of plasmid DNA will be determined by the efficiency of transfection , which is very dependent upon the particular cell line and transfection protocol. - [Read B- Galactosidase Assay Protocol]

Calcium Phosphate Transfection Method - http://www.flemingtonlab.com/Protocols/CalciumPhosphateTransf.pdf

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This calcium phosphate transfection method works best in cell lines that are 1) highly transformed and 2) adherent (Hela, U2OS, SAOS2, AdAH, NPC-KT and obtain from 20% to 100% transfection efficiency depending on the cell line). Works well for transient experiments but precautions should be used in the design and interpretation of experiments based on the discussion below. Also works very well for generating stable cell lines. This method is quite sensitive to the amount of input plasmid. - [Read Calcium Phosphate Transfection Method]

cDNA Synthesis and Cloning - http://omrf.ouhsc.edu/~frank/CDNA.html

Category: RNA Protocols: cDNA Libraries Library

Protocol for cDNA synthesis and cloning cDNA into plasmid vector. 1st Strand cDNA Synthesis, and determine the efficiency of first strand cDNA synthesis. Also includes second Strand cDNA Synthesis. Dr.Frank - [Read cDNA Synthesis and Cloning]

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]

Cleavage Efficiency Close to the Termini of PCR Fragments - http://www.fermentas.com/techinfo/re/restrdigpcrii.htm

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Cleavage Efficiency Close to the Termini of PCR Fragments. List of restriction enzymes and their cleavage efficiencies near the ends of DNA. Fermentas - [Read Cleavage Efficiency Close to the Termini of PCR Fragments]

Competitive RT-PCR: Estimation of Copy Number Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4111

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment. A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor. The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Competitive RT-PCR: Preparation of Competitor RNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4110

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor. This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest. Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Determining Plating Efficiency in Yeast: Direct Method - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4185

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Direct method for determining efficiency in yeast. The plating efficiency of a strain is a measure of the percentage of cells in a culture that are capable of forming colonies (colony forming . - [Read Determining Plating Efficiency in Yeast: Direct Method]

Determining Plating Efficiency in Yeast: Indirect Method Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4184

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Protocol for the indirect method of determining plating efficiency in yeast. - [Read Determining Plating Efficiency in Yeast: Indirect Method Protocol]

DNA Transfection Using Polybrene Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3875

Category: Transfection Protocols

Polybrene and DMSO can be used to achieve stable transformation of several types of cells by plasmid DNA. The yield of transformants is up to 15-fold greater with Polybrene than with calcium phosphate-DNA coprecipitation. However, there is no difference between the two methods in the efficiency of transformation of cells by high-molecular-weight DNA. - [Read DNA Transfection Using Polybrene Protocol]

Hepatoma Cell Cultures as In Vitro Models for Hepatotoxicity - http://embryo.ib.amwaw.edu.pl/invittox/prot/13.htm

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

This test is designed to detect irreversible toxic effects on both cell growth and survival, by the evaluation of colony-forming (CF) efficiency, in hepatoma cell lines derived from man, rat and mouse. - [Read Hepatoma Cell Cultures as In Vitro Models for Hepatotoxicity]

High Efficiency FCC Preparation and Tx Protocol - http://genetics.med.harvard.edu/~cepko/protocol/mike/F4.html

Category: Competent Cells Protocols

This is an easy and straightforward protocol that gives efficiencies of 106 to 107 cfu/mg of plasmid DNA. A growth curve is required for each strain that is prepared.
- [Read High Efficiency FCC Preparation and Tx Protocol]

High Efficiency LiAc Transformation Protocol - http://www.fhcrc.org/science/labs/gottschling/yeast/ytrans.html

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high efficiency LiAc transformation. - [Read High Efficiency LiAc Transformation Protocol]

High Efficiency Transformation Protocol - http://www.umanitoba.ca/faculties/medicine/biochem/gietz/method.html

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high efficiency transformation. - [Read High Efficiency Transformation Protocol]

High-Efficiency LiOAc Transformation of S. cerevisiae Protocol - http://www.biochem.emory.edu/labs/acorbe2/yeasttransformation.html

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high-efficiency LiOAc transformation of S. cerevisiae. - [Read High-Efficiency LiOAc Transformation of S. cerevisiae Protocol]

High-Efficiency Transformation of Yeast Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4145

Category: Yeast Protocols: Yeast Transformation Protocols: High Efficiency Yeast Transformation Protocols

Protocol for high-efficiency transformation of Yeast. - [Read High-Efficiency Transformation of Yeast Protocol]

Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4317

Category: C. elegans Protocols: C. elegans Genetics Protocols

Protocol describes an easily scalable way of introducing double-stranded RNA (dsRNA) in Caenorhabditis elegans: feeding the nematode with bacteria that express dsRNA. When using an RNase-III-negative Escherichia coli strain (HT115), the efficiency of this method is comparable to the alternative. - [Read Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol]

Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4317

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol describes an easily scalable way of introducing double-stranded RNA (dsRNA) in Caenorhabditis elegans: feeding the nematode with bacteria that express dsRNA. When using an Rnase-III-negative Escherichia coli strain (HT115), the efficiency of this method is comparable to the alternative. - [Read Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol]

Lipofectin Increases the Efficiency of DNA-Mediated Transformation of Neurospora Crassa - http://www.fgsc.net/fgn38/selitren.html

Category: Fungi Protocols: Neurospora Protocols

Lipofectin increases the efficiency of DNA-mediated transformation of Neurospora crassa. - [Read Lipofectin Increases the Efficiency of DNA-Mediated Transformation of Neurospora Crassa]

Molecular Cloning of PCR Products Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=3DC8DA9CD690052F2234D07559CD9BFE&objectid=6676A195E9797060CC491B4B58ECC2E1

Category: PCR Protocols: PCR Cloning Protocols

The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This protocol describes the strategies for generating and manipulating suitable ends on the PCR fragments. - [Read Molecular Cloning of PCR Products Protocol]

Oligofection of Small Interfering RNAs in Senescent Human Fibroblasts Protocol - http://www.natureprotocols.com/2006/12/06/oligofection_of_small_interfer.php

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol for oligofection of small interfering RNAs in senescent human fibroblasts. This protocol allows the transfection of human senescent cells (regardless of the cause of cellular senescence) with an efficiency of around 70%. - [Read Oligofection of Small Interfering RNAs in Senescent Human Fibroblasts Protocol]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. Cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. Regardless of cell size or phenotype, transfection efficiency increases with a high concentration of cells in a small volume. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes transfection of plasmid DNA into mammaliancell lines using electroporation, a process whereby externalapplication of electric pulses induce cell membrane permeability.A number of factors can affect electrotransfection efficiency.In general, cells in suspension and small volume cells are difficultto transfect, whereas adherent cells and large volume cellsare relatively easy. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

PEI Nanoparticles for Targeted Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4451

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection. - [Read PEI Nanoparticles for Targeted Gene Delivery Protocol]

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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.

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.

Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.

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