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Topic: PCR

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	The Polymerase Chain Reaction (Site not responding. Last check: 2007-10-20)
		For PCR, primers must be duplicates of nucleotide sequences on either side of the piece of DNA of interest, which means that the exact order of the primers' nucleotides must already be known.
		PCR looks directly for the virus's unique DNA, instead of the method employed by the standard test, which looks for indirect evidence that the virus is present by searching for antibodies the body has made against it.
		PCR can provide enormous peace of mind to people who are trying to have children- for example, by reassuring anxious parents-to-be that they run no risk of having a child with a particular genetic disease.
	www.faseb.org /opar/bloodsupply/pcr.html (3752 words)

	Encyclopedia: SARS
		The last test is a PCR (polymerase chain reaction) test that can detect genetic material of the SARS virus in specimens ranging from blood, sputum, tissue samples and stool.
		This means that while a positive PCR test result is strongly indicative that the patient is infected with SARS, a negative test result does not mean that the patient does not have SARS.
		On electron microscopy, these tissue viral inclusions resembled coronaviruses, and comparison of viral genetic material obtained by PCR with existing genetic libraries suggested that the virus was a previously unrecognized coronavirus.
	www.nationmaster.com /encyclopedia/SARS (4443 words)

	BCH5425 Molecular Biology and Biotechnology (Site not responding. Last check: 2007-10-20)
		PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence.
		PCR amplification is achieved by using oligonucleotide primers.
		PCR was invented in 1985 by Kary Mullis, working for Cetus corporation somewhere near Berkeley, California.
	wine1.sb.fsu.edu /bch5425/lect22/lect22.htm (1151 words)

	PCR (Site not responding. Last check: 2007-10-20)
		In the polymerase chain reaction (PCR), a thermostable DNA polymerase amplifies DNA that is flanked by known sequences.
		PCR can be used in many complex ways to achieve different results.
		The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes.
	omrf.ouhsc.edu /%7Efrank/PCR.html (1932 words)

	RT-PCR
		It is a technique in which an RNA strand is transcribed into a DNA complement to be able to subject it to amplification by polymerase chain reactions (PCR).
		Transcribing an RNA strand into a DNA complement is termed reverse transcription (RT) and is done by the enzyme reverse transcriptase.
		To avoid confusion with the 2 "RT", the term quantitative PCR or quantitative RT-PCR is preferred.
	encycl.opentopia.com /term/RT-PCR (198 words)

	Real Time PCR Primer Sets !!!
		As with all PCR amplifications, however, the specific reaction conditions for each set must be optimized, particularly primer concentration, annealing temperature and magnesium chloride concentration.
		PCR products may be quantitated by generating a standard curve or quantitated relative to a control gene.
		One must be sure, however, that the efficiency of PCR is the same for the standards as that of the "unknown" samples.
	www.realtimeprimers.org (936 words)

	What the Heck is PCR?
		Polymerase chain reaction (PCR) is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
		In order to use PCR, one must already know the exact sequences which flank (lie on either side of) both ends of a given region of interest in DNA (may be a gene or any sequence).
		The first step for PCR would be to synthesize "primers" of about 20 letters-long, using each of the 4 letters, and a machine which can link the letters together in the order desired - this step is easily done, by adding one letter-at-a-time to the machine (DNA synthesizer).
	people.ku.edu /~jbrown/pcr.html (866 words)

	[No title]
		PCR primers performed well shortly after being purchased, but after storage for several months as diluted stocks in water within a non-frost-free -20 degrees C freezer, these same primers failed to give the same amount of amplified product in identical reactions.
		Roger Aeschbacher (aeschba@fmi.ch) suggested that some primer sets cause PCR failure by nonspecific priming because they are more likely to form primer dimers or intrastrand secondary structures, such as hairpins, only after undergoing multiple cycles of warming and slow freezing.
		This is accomplished by withholding a critical component from the reaction mixture, and is usually performed by separating the template and primers from the buffer components and thermostable polymerase with a physical barrier of wax.
	www-lecb.ncifcrf.gov /~pnh/papers/TIBS/jan95.html (1407 words)

	TechNotes 8(1): Real-time PCR Goes Prime Time
		Real-time PCR assays used for quantitative RT-PCR combine the best attributes of both relative and competitive (end-point) RT-PCR in that they are accurate, precise, capable of high throughput, and relatively easy to perform.
		During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5' exonuclease activity of the polymerase cleaves the probe.
		Real-time PCR machines are not cheap, currently about $60-$95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis.
	www.ambion.com /techlib/tn/81/813.html (1323 words)

	TB #176: DNA Contamination in RT-PCR
		PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination.
		Thus, the size of a PCR product amplified from a pseudogene may be identical to that produced from a cDNA copy.
		The lanes to the left of the markers are PCR reactions done without reverse transcription, demostrating the lack of genomic DNA contamination in these RNA samples.
	www.ambion.com /techlib/tb/tb_176.html (1785 words)

	Principle of the PCR (Site not responding. Last check: 2007-10-20)
		The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene.
		Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene.
		The ladder is a mixture of fragments with known size to compare with the PCR fragments.
	allserv.rug.ac.be /~avierstr/principles/pcr.html (606 words)

	Pinning down PCR
		In reverse-transcriptase PCR (RT-PCR), which begins with RNA instead of DNA, the efficiencies of reverse transcription for the template and the standard also might be different.
		Another drawback to competitive PCR is low throughput, says Carl Wittwer, a University of Utah researcher who developed a fluorescent instrument for quantitative PCR.
		Exonuclease and hybridization methods for real-time PCR are well established, but there are other options, including hairpin probes and hairpin primers, which are named for the way they fold back on themselves.
	pubs.acs.org /hotartcl/ac/99/mar/pcr.html (3436 words)

	Disruption by Fusion PCR
		PCR amplify the marker using the m13 forward and reverse primers.
		The Prakash and Jones vectors are useful for the marker PCR.
		Note: If PCR generated errors are of concern then each PCR generated fragment can be extracted from the low melt agarose and EtOH precipitated.
	www.upstate.edu /biochem/amberg/protocols/fusion_PCR.html (281 words)

	The Microinjection Workshop
		PCR of blood, hair or small tissue samples
		Note: It has been reported that PCR of blood is NOT possible when the blood component of the reaction mixture exceeds 1%.
		After amplification, 10 ul of PCR product can be visualized on a gel.
	ourworld.compuserve.com /homepages/TheBroons/tnp8.htm (316 words)

	PCR Protocols
		PCR Protocols in Molecular Toxicology is a practical guide to the use of polymerase chain reaction (PCR) to help examine, on a molecular and cellular level, how toxic responses are manifested.
		Make sure that any PCR machine you buy is fully programmable (can do the cycle profile as well as certain advanced options such as "touch-up" or "touch-down" and increasing elongation time/cycle), can handle 96-well plates and has a heated lid for oil-free operation.
		Although PCR is considered science, (it is simply a glorified enzyme assay), there is a lot of art involved and a little luck never hurts either.
	www.cas.psu.edu /docs/CASDEPT/VET/jackvh/jvhpcr.html (5958 words)

	protocol for competitive RT-PCR
		To make a standard, first a PCR with a conventional downstream primer and a modified upstream primer (40 nucleotides in length) is performed according to Celi et al.
		In the following PCR 3-5 µl of cDNA, 1.5 units Taq DNA polymerase (Pharmacia), 200 µM of each dNTP, 250 nM of each primer and 1/10 volume of a 10x PCR standard buffer (15 mM MgCl2; 100 mM Tris/HCl, pH 8.3; 500 mM KCl) are added to a total volume of 50 µl.
		The amount of cDNA used for a PCR, the number of cycles and the nucleic acid stain used depend on how abundant the transcript is that is being measured.
	www.uni-ulm.de /~bbartelt/britta/protokoll/rt-pcr.html (995 words)

	Real-Time PCR [M.Tevfik DORAK]
		Real-time PCR does not detect the size of the amplicon and thus does not allow the differentiation between DNA and cDNA amplification, however, it is not influenced by non-specific amplification unless SYBR Green is used (see below).
		By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template.
		The downstream PCR primer also acts as the primer for reverse transcriptase (random hexamers or oligo-dT cannot be used for reverse transcription in one-step RT-PCR).
	dorakmt.tripod.com /genetics/realtime.html (6378 words)

	BDGP: Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation
		PCR Set up PCR reactions with primers appropriate for the type of P element and the end of the element from which you want to recover genomic sequence (see table below and Figures).
		Examine 5 ul of the PCR reactions on 1.5% agarose gel.
		The ALF sequencers are apparently more sensitive to excessively strong signals therefore the amount of PCR product added to the cycle sequencing reactions may need to be adjusted, especially in the case of very small products, in order to obtain acceptable sequence reads.
	www.fruitfly.org /about/methods/inverse.pcr.html (927 words)

	Long PCR Protocol
		Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration.
		The constant one minute is probably necessary for primer annealing/extension to occur; at 68 degrees C the kinetics of primer-template annealing and melting may become the limiting factor in the rate of primer extension.
		I put the template/primer fraction in the tube and heat in the PCR machine to 94 degrees C for 10 sec.
	twod.med.harvard.edu /labgc/estep/longPCR_protocol.html (725 words)

	Rational primer design greatly improves differential display-PCR (DD- PCR) -- Graf et al. 25 (11): 2239 -- Nucleic ...
		Recombinant clones were screened by PCR, plasmid DNA was prepared and sequenced.
		Primers and cycling conditions in DD-PCR protocols were originally designed such that 100-150 different primer combinations encompass most of the estimated 15 000 expressed genes (2,3).
		A reason for this might be that the competitive nature of the underlying PCR was not fully appreciated.
	nar.oupjournals.org /cgi/content/full/25/11/2239 (1527 words)

	Polymerase Chain Reaction
		PCR, like DNA sequencing, is based on the DNA polymerization reaction.
		The main difference with PCR is that, in addition to using a primer that sits on the 5' end of the gene and makes a new strand in that direction, a primer is made to the opposite strand to go in the other direction.
		The powerful amplification of PCR may be able to detect this cross contamination of samples.
	faculty.plattsburgh.edu /donald.slish/PCR.html (1224 words)

	20-mer Polymerase Chain Reaction Procedure (for MJ Research Thermal Cycler)
		While the PCR cycle is processing at Td=94 C, 30 sec (1st stage and 1st of 45 cycle), open the top of MFTs.
		When the transition stage starts (between Td and Ta) of the 1st cycle, pause the cycle (push a decimal point button) and add a mixture of Taq polymerase (2.8 µl/tube) with EDP (25 µl liquid end) as quick as possible.
		GeneAmp 9600(Perkin Elmer Cetus): 10-mer PCR for amplification of random genomic DNA fragments of Cotton
	wheat.pw.usda.gov /homepage/lazo/methods/lazo/pcrproto.html (1001 words)

	Degenerate PCR a short guide
		Degenerate PCR is in most respect identical to ordinary PCR, but with one major difference.
		Instead of using specific PCR primers with a given sequence, you use mixed PCR primers.
		This means that the first PCR cycles are very inefficient, and you some times have to run 50 cycles PCR just to see a faint band of your gene.
	boneslab.chembio.ntnu.no /DegPCRshortguide.html (1392 words)

	Phenol Extraction
		Separate the PCR reaction products by electrophoresis in a TAE agarose gel containing ethidium bromide using standard protocols.
		For each completed PCR reaction, transfer the aqueous (lower) phase to a clean microcentrifuge tube.
		The presence of too much mineral oil in the sample can lead to a decreased yield in the PCR product purification.
	www.research.umbc.edu /%7Ejwolf/m8.htm (637 words)

	University of Iowa DNA Facility - "Real-Time" PCR
		Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5' end and the quencher at the 3' end.
		Additional reporter dye molecules are cleaved from their respective probes with each cycle, effecting an increase in fluorescence intensity proportional to the amount of amplicon produced.
		Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles.
	dna-9.int-med.uiowa.edu /realtime.htm (2248 words)

	Standard PCR
		PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790).
		It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.
		However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al.
	www.uct.ac.za /microbiology/pcrcond.htm (1789 words)

	Sizing Up Real-Time PCR - Biocompare Technology Spotlight
		One of the more recent adaptations of PCR is Real-Time PCR, a technique that allows researchers to quantify the amount of DNA or RNA in a given sample.
		The underlying principle behind Real-Time PCR is fairly straightforward — by measuring the amount of PCR product created during the exponential phase of amplification one can determine the amount of starting material.
		As the PCR reaction proceeded, it produced double-stranded DNA, which bound ethidium bromide, resulting in an increase in fluorescence.
	www.biocompare.com /spotlight.asp?id=37 (514 words)

	Effect of Primer Purity on the Banding Patterns of Differential Display Polymerase Chain Reaction
		In the case of primers for sequencing or PCR amplifications, a crude reaction mixture may be used immediately following its synthesis, cleavage, and deprotection.
		The PCR cycling profile was 94°C for 5 minutes (94°C for 30 seconds, 42°C for 1 minute, 72°C for 30 seconds) for 40 cycles and then 72°C for 5 minutes.
		The 17 different PCR fragments previously identified as being differentially expressed in normal and IL-2-stimulated T1 cell lines were excised and reamplified with the same primer used in the original DD-PCR reactions.
	www.abrf.org /JBT/2000/june00/jun00wen.html (2442 words)

	Tavi's PCR protocols
		No parts of this work (text, tables or pictures) may be commercialized, published or otherwise reproduced without the written consent of the author.
		For a complete description of primers, PCR programs and a discussion of the PCR conditions please consult: Andrologia 26: 97-106 (1994) and Biotechniques 23: 504-511 (1997).
		PCR guide: a discussion of the main parameters influencing the outcome of the PCR and multiplex PCR reaction in 16 pages/sections and using over 45 pictures
	info.med.yale.edu /genetics/ward/tavi/PCR.html (155 words)

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