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Category Matches: 27
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- Quantitative RT-PCR and Other PCR Procedures (Jack Vanden Heuvel, Penn State University)
A must-read protocol for PCR beginners as well as verterans. It touches in depth aspects specific to quantitative RT-PCR as well as those common to standard PCR.
http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvh...
Added: Sun Mar 28 2004, Hits: 8604, Reviews: 0 Write review -
Essentials of Real-Time PCR (Life Technologies)
Things you must know before you do qPCR including basics about Real-Time PCR Assays and Sequence Detection Chemistries SYBRŽ Green I Dye Chemistry About Quantitation Assays Absolute vs. Relative Quantitation Standard Curve Method for Relative Quantitation Comparative CT method for Relative Quantitation Standard Curve Method for Absolute Quantitation
http://www.invitrogen.com/site/us/en/home/Products...
Added: Thu May 02 2013, Hits: 4383, Reviews: 0 Write review Cached -
The PCR Suite
(Van Baren MJ, Heutink P, Erasmus Medical Center)
a collection of programs that interact with the Whitehead's Primer3 program. The original Primer3 program designs primersets for one sequence and it allows you to change almost every parameter. The PCR Suite has more stringent settings (to allow more working primersets) and a smaller range of parameters (because you won't change most of them anyway). The suite consists of four programs: Overlapping_Primers is for creating multiple overlapping PCR products in one sequence. Genomic_Primers designs primers around exons in genomic sequence. All you need is a GenBank file containing your gene. SNP_Primers designs primers around every SNP in a GenBank file. cDNA_Primers designs primers around open reading frames. Simply upload a GenBank file containing your genes.
http://www.eur.nl/fgg/kgen/primer/index.html
Added: Tue Mar 02 2004, Hits: 3030, Reviews: 0 Write review Cached -
Cloning of PCR Products with Phosphorylated Primers
(Hideaki Shiraishi, Kyoto University)
For cloning DNA fragments by ligating with dephosphorylated vector DNA, the fragments should have phosphates on their 5' termini. Since phosphorylation of PCR products by T4 polynucleotide kinase is inefficient, primers should be phosphorylated prior to PCR reaction.
http://kuchem.kyoto-u.ac.jp/seika/shiraishi/protoc...
Added: Fri Jan 30 2004, Hits: 3086, Reviews: 0 Write review Cached -
Colony PCR ( Cindy Troxell)
Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from non-homologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting, but an adjunct.
http://www-rcf.usc.edu/~forsburg/pcr.html
Added: Tue May 14 2002, Hits: 4506, Reviews: 0 Write review Cached -
Thermal Cycling Profile for Standard PCR (Roche Diagnostics Corporation)
Basic guide to PCR cycling and how to optimize each step.
http://www.roche-applied-science.com/pcr/applicati...
Added: Sat Sep 21 2002, Hits: 591, Reviews: 0 Write review Cached - Step by Step SSCP
(Travis Glenn, Laboratory of Molecular Systematics, Smithsonian Institution)
This protocol is for Single-Strand Conformation Polymorphism (SSCP) analysis. It is basically the same as any SSCP protocol you will find in the literature. The only major difference is that the author recommends doing a two reaction PCR (unless you are screening more than 48 individuals, then you should optimize PCR conditions and amplify hot the first time around). The first reaction is a "normal" cold PCR to amplify the intended products. Check that these reactions were successful by running out on a minigel. The second reaction is a hot PCR to label the products produced in the first PCR reaction. The hot product will be diluted in sequencing buffer, denatured, and loaded onto a native polyacrylamide gel.
http://www.uga.edu/srel/DNA_Lab/SSCP'96V2.rtf
Added: Mon Sep 23 2002, Hits: 1426, Reviews: 0 Write review Cached - SYBR Green Quantitative PCR Protocol (Lusis Lab, UCLA)
Quantitative PCR is a method used to detect relative or absolute gene expression level. All qPCR involves the use of fluorescence to detect the threshold cycle (Ct) during PCR when the level of fluorescence gives signal over the background and is in the linear portion of the amplified curve. This Ct value is responsible for the accurate quantization of qPCR.
http://www.genetics.ucla.edu/labs/lusis/greenquant...
Added: Sat Mar 21 2009, Hits: 31957, Reviews: 0 Write review Cached -
Single Primer (Semi-Random) PCR (John Roth, Division of Biological Sciences,University of California at Davis)
Single primer PCR allows amplification from known to unknown regions in chromosomes, phage, plasmids, large PCR products and other sources of DNA.
http://rothlab.ucdavis.edu/protocols/semirandom.ht...
Added: Tue May 25 2004, Hits: 793, Reviews: 0 Write review Cached -
Genotyping Transgenic Mice by PCR (University of Michigan Transgenic Animal Model Core)
Test mice for the presence of the transgene by PCR. It is provided for those investigators who are unfamiliar with mouse genotyping and who need to establish a genotyping method.
http://www.med.umich.edu/tamc/pcr.html
Added: Tue May 14 2002, Hits: 1267, Reviews: 0 Write review Cached -
RT In Situ PCR (Gerard J. Nuovo)
RT in situ PCR allows for the routine and rapid detection of low copy viral and human RNAs. Success with RT in situ PCR is best accomplished with formalin fixed, paraffin embedded material, which allows the study of archival material.
http://www.bioscience.org/1996/v1/c/nuovo1/htmls/l...
Added: Tue May 14 2002, Hits: 2261, Reviews: 0 Write review Cached - Standard RT-PCR (Pamela Stanley Lab Wiki, Yale University)
RT-PCR or reverse transcription PCR refers to PCR that uses product of an RT reaction as template. In effect, the PCR amplifies cDNA fragments. In one-tube RT-PCR, RNA and PCR primers are added to a reaction mix that is thermocycled for RT first followed by for PCR. One-tube RT-PCR reaction mixes are supplied by many manufacturers. Drawbacks to their use include lack of flexibility. Otherwise, RT-PCR is a two step process.
http://stanxterm.aecom.yu.edu/wiki/index.php?page=...
Added: Fri Mar 20 2009, Hits: 18064, Reviews: 0 Write review Cached - PCR Additives (Rob Cruickshank, University of Glasgow)
A variety of PCR additives and enhancing agents have been used to increase the yield, specificity and consistency of PCR reactions.
http://www.uni-mainz.de/~lieb/additiva.html
Added: Thu Apr 22 2004, Hits: 6702, Reviews: 0 Write review Cached - PCR and Agarose Gel Protocol
(Genomics Facility, University of Edinburgh)
Describes PCR reaction and running agarose gel
http://staff.biology.ed.ac.uk/services/equipment/g...
Added: Sat Jan 31 2009, Hits: 4856, Reviews: 0 Write review Cached -
Standard PCR (Chen)
Standard PCR, primer design, cycling, additives and more
http://www.biochem.ucl.ac.uk/~chen/protocols/PCR.h...
Added: Tue May 14 2002, Hits: 5738, Reviews: 0 Write review - PCR from Plant Tissue
(Schneitz Lab, University of Zurich)
Use plant such as leaves or flowers tissue directly for PCR without DNA extraction
http://www.unizh.ch/botinst/Cyto_Website/schneitzL...
Added: Tue May 14 2002, Hits: 2661, Reviews: 0 Write review Cached -
Preparation of Alpha-satellite DNA by PCR
(Thomas Ried's Lab, NCI)
Detailed procedures including Primary PCR for amplification of alpha satellite sequences, Secondary PCR for labeling of alpha satellite sequences, FISH hybridizations of alpha satellite DNA probes.
http://www.riedlab.nci.nih.gov/publications/2443.3...
Added: Fri Oct 18 2002, Hits: 680, Reviews: 0 Write review - Addition of 3'-A Overhangs (A-Tailing) to PCR Product (Protocol Online)
PCR product amplified by proofreading polymerases is blunt-ended because proofreading polymerases possess 3´→5´ exonuclease activity that removes the 3´-A overhangs necessary for TA cloning. However, 3´-A overhangs can be added to blunt-end fragments using a Taq polymerase after PCR amplification.
Added: Mon Feb 02 2009, Reviews: 0 Write review - Real-Time PCR (M.Tevfik Dorak, MD, PhD)
Excellent guide to real-time PCR including background; TaqMan primer and probe design guidelines; General recommendations for real-time RT-PCR;Setting up one-step TaqMan reaction;Cycling parameters for one-step PCR;Interpretation of results; The comparative CT method (DDCT) for relative quantitation of gene expression; Points to remember and trouble shooting; and more... (alternative url: http://home.att.net/~dorak/genetics/realtime.html)
http://dorakmt.tripod.com/genetics/realtime.html
Added: Sun Sep 22 2002, Hits: 39270, Reviews: 0 Write review Cached - PCR Mutagenesis Protocol (Michael Bumbulis, Baldwin-Wallace College)
PCR is based on the enzymatic amplification of a DNA fragment that is flanked by two oligonucleotide primers that anneal (stick) to opposite strands of the target sequence. The primers are oriented with their 3' ends pointing towards each other (recall that DNA is synthesized in the 5' to 3' direction). These primers are then extended by the polymerase enzyme. Repeated cycles of heat denaturation, annealing of the primers to their complementary sequences and extension of the primers result in the amplification of the segment defined by the 5' ends of the primers...
http://homepages.bw.edu/~mbumbuli/molbio/labs/pcr/...
Added: Mon Feb 18 2013, Hits: 2358, Reviews: 0 Write review Cached - PCR reaction components
(Roche Diagnostics Corporation)
Very useful introduction to PCR componets including template, primers, dNTP, additive, polymerase, pH, and MgCl2 concentration.
http://www.roche-applied-science.com/pcr/applicati...
Added: Sat Sep 21 2002, Hits: 770, Reviews: 0 Write review Cached - PCR Additives
(OpenWetWare)
A complete rundown of PCR additives and their roles in PCR reaction
http://openwetware.org/wiki/Berglund:PCR_Additives
Added: Sun Mar 15 2009, Hits: 5748, Reviews: 0 Write review - PCR Primer Design Guide
This tutorial describes the most fundmental aspects of PCR primer design and PCR reaction including theories and applications (PPT slides).
http://www.biotechlab.nwu.edu/pe/
Added: Tue Oct 21 2003, Hits: 1662, Reviews: 0 Write review Cached - PCR (Fermentas)
Gives detailed general protocol for PCR, including preparing reaction mixture, cycling conditions, guidelines to avoid contamination and more...
http://www.fermentas.com/techinfo/PCR/dnaamplproto...
Added: Tue May 14 2002, Hits: 17085, Reviews: 0 Write review Cached -
Standard PCR (Biotech)
Detailed PCR protocol including preparation of the sample, the master mix and the primers, detection and analysis of the reaction products.
http://www.accessexcellence.org/LC/SS/PS/PCR/PCR_t...
Added: Tue May 14 2002, Hits: 9235, Reviews: 0 Write review Cached