This month in Alkami PCR Reviews, Lance Larka talks about free online primer design software. Featured in June are DOPE2, DoPrimer, NetPrimer, and Oligos-U-Like/Primers3. His article is at: http://www.alkami.com/reviews/rvwdsgn3.htm Our current list of primer tool links and brief descriptions that we have at http://www.alkami.com/primers/refdsgn.htm includes: * CODEHOP * DOPE2 * DoPrimer * The GPRIME Package * NetPrimer * Oligos-U-Like * Primer Design * Primer3 * Primers! * Primers! Lite * Primer Selection * STS Pipeline v1.2 * The Primer Generator * Web Primer (Stanford) * Web Primers * WWW GeneFisher * xprimer Other primer design resources are listed at http://www.alkami.com/primers/idxprmr.htm The Alkami Biosystems site, http://www.alkami.com, is a comprehensive resource for the polymerase chain reaction. We have online references that include primer design tips and tools, reagent concentrations, polymerase characteristics, PCR methods, and temperature calculations. In addition, you will find ...
Primer Design for PCR: Primer guidelines page offers a look at the general and useful guidelines laid for designing primers for PCR reaction including Primer Tm considerations, PCR primer cross dimer values, annealing temperature and primer GC%
In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3 end of primed double stranded DNA, the binding of the 3 end of the primer is especially important, while the 5 end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3 to the 5 end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3 base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3 to 5 order to fabricate the ...
In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3 end of primed double stranded DNA, the binding of the 3 end of the primer is especially important, while the 5 end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3 to the 5 end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3 base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3 to 5 order to fabricate the ...
Download Primer3 - PCR primer design tool for free. Design PCR primers from DNA sequence. Widely used (190k Google hits for primer3).
Dear All, I searched for an 18S rRNA gene of an organism on the NCBI website. I found the sequence and designed primers using the primer tool on the same website. Now, I want to check the primer specificity (i.e. can I use this primer as an identification tool to detect a specific species using PCR) and the question is: do I need to BLAST the primer sequence (both forward and reverse) or to BLAST the sequence of the amplified fragment (PCR product). Many thanks in advance. Best regards Mohammad Email: moh_aldeeb from yahoo.com Personal Website: http://sites.google.com/site/draldeebsite/ --- On Wed, 7/6/11, mnr mnr ,mnr475 from gmail.com, wrote: , From: mnr mnr ,mnr475 from gmail.com, , Subject: Blunt end cloning , To: methods from magpie.bio.indiana.edu , Date: Wednesday, July 6, 2011, 1:23 PM , Hi all. , , I have tried cloning a potential toxic gene about 1.2 kbp , into E.coli with , T7 promoter. After two months, I am still not successful. I , am now plannig , to do cloning into pUC18 vector ...
This was important enough that I wanted to get it out immediately. My research into the NCBI database for nucleotide sequences has lead to a stunning discovery. One of the WHO primer sequences in the PCR test for SARS-CoV-2 is found in all human DNA!. The sequence CTCCCTTTGTTGTGTTGT is an 18-character primer sequence found in the WHO coronavirus PCR testing protocol document. The primer sequences are what get amplified by the PCR process in order to be detected and designated a positive test result. It just so happens this exact same 18-character sequence, verbatim, is also found on Homo sapiens chromosome 8! As far as I can tell, this means that the WHO test kits should find a positive result in all humans. Can anyone explain this otherwise?. I really cannot overstate the significance of this finding. At minimum, it should have a notable impact on test results.. ...
(KudoZ) English to Czech translation of Oligos/Primers/Probes: Oligonukleotidy / primery / sondy [Biochemie - Chemistry; Chem Sci/Eng (Science)].
Sequencing Primers and Plasmids - posted in Molecular Biology: Im a little confused about the notation of the sequencing primers listed on the plasmid map for Invitrogens pCR 2.1 and 4.0 used in TOPO TA cloning. Why is the forward primer 3 insertion site while the reverse primer 5 insertion. I thought it is opposite of that. Looking for some explanation. Wondering if Im looking at the map wrong, or if Im not understanding something. Link for map is below.http:...
A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5 terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the ohgonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated. ...
The sequence CTCCCTTTGTTGTGTTGT is an 18-character primer sequence found in the WHO coronavirus PCR testing protocol document. The primer sequences are what get amplified by the PCR process in order to be detected and designated a positive test result. It just so happens this exact same 18-character sequence, verbatim, is also found on Homo sapiens chromosome 8! As far as I can tell, this means that the WHO test kits should find a positive result in all humans. Can anyone explain this otherwise ...
GENEWIZ offers a variety of free universal primers for sequencing. These free universal primers are being updated to reflect the needs of our customers. Users in our new CLIMS Online Ordering and Data Management System have access to the Updated GENEWIZ Universal Primer list (see below). If you are unsure which CLIMS system you are using, please contact our Technical Support team at 877-GENEWIZ, ext. 2. ...
OLIGO performs a range of functions for researchers in PCR and related technologies, enables to design consensus, multiplex and degenerate primers. Reverse translation, restriction enzyme and open reading frames analysis, oligonucleotide database, primer secondary structure, LCR, siRNA, molecular beacons and nested primers design, real time PCR, batch file processing.
The reasons for primer dimer formation in an NTC are often due to multiple factors. These include: sub-optimal primer annealing temperature (often due to differences in buffering conditions between different qPCR kits), sub-optimal primer synthesis (HPLC-purified primers result in less primer dimer formation and are useful for low copy number detection), and poor primer design (using software such as Primer3 is recommended when designing primers). An alternative is to reduce the total number of cycles in a qPCR reaction if amplification of the primer dimer lies outside of the range of the experimental data eg. if the sample being tested has an average Cq of 28 cycles and contains specific product only (no primer dimer), and the NTC amplifies a primer dimer after 37 cycles, the reaction can be run for 35 cycles, rather than 40. This approach can be taken only when the sample being tested gives rise to a specific product and not a combination of specific product and primer dimer, and also when the ...
RT-PCR primer design - posted in PCR, RT-PCR and Real-Time PCR: Hi, I want to ask about how to design primer for RT-PCR. Specifically, i want to design primer based on the human mRNA encoding the c-myc, p53, caspase-3 and bcl-2 gene. I have no clue where to start and what to do... thank you..
( http://www.abnova.com ) - Primer-BLAST was developed to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST...
Gel Layout:. Lane 1 - Hyperladder 1. Lanes 2-6 = 5′ RACE Library. Lane 2 - nGSP1 (5′ RACE primer). Lane 3 - nGSP2 (3′ RACE primer). Lane 4 - Neg. Control (no RACE primers). Lane 5 - Neg. Control (nGSP1, no Universal primer). Lane 6 - Neg. Control (nGSP2, no Universal primer). Lane 7 - Empty. Lanes 8-12 = 3′ RACE Library. Lane 8 - nGSP1 (5′ RACE primer). Lane 9 - nGSP2 (3′ RACE primer). Lane 10 - Neg. Control (no RACE primers). Lane 11 - Neg. Control (nGSP1, no Universal primer). Lane 12 - Neg. Control (nGSP2, no Universal primer). First of all, we see the appropriate response of each primer only producing amplicons in their respective libraries (i.e. 5′ primer only works in 5′ RACE library). This simply confirms that the primers were designed correctly. Secondly, our negative controls are clean. Thirdly, we get distinct bands from both primers. The bands marked with blue arrows in the image above were excised and purified using Ultrafree DA spin columns (Millipore). These ...
Methylated/Non-methylated Control DNA & Primer Set DNA ZD5101-2 Methylated/Non-methylated Control DNA & Primer Set DNA ZD5101-2
Now that my lab is fully equipped, Im taking on rotation students. Unfortunately, with the pandemic, its harder to have one-on-one meetings where I can sit down and walk the new students through every method. Furthermore, why repeat teaching the same thing to multiple students when I can just make an initial written record that everyone can reference and just ask me questions about? Thus, heres my instructional tutorial on how I design primers in the lab.. First, its good to start out by making a new benchling file for whatever youre trying to engineer. If youre just making a missense mutation, then you can start out by copying the map for the plasmid youre going to use as a template. Today, well be mutating a plasmid called G619C_AttB_hTrim-hCPSF6(301-358)-IRES-mCherry-P2A-PuroR to encode the F321N mutation the CPSF6 region. This should abrogate the binding of this peptide to the HIV capsid protein. Eventually every plasmid in the lab gets a unique identifier based on the order it ...
Pick PCR primers and hybridization oligos Version: EMBOSS:6.6.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall The sequence from which to choose primers. The sequence must be presented 5 to 3 [-outfile] outfile [*.eprimer32] Whitehead primer3_core program output file Additional (Optional) qualifiers (* if not always prompted): -[no]primer toggle [Y] Tell Eprimer32 to pick primer(s) * -task menu [1] Tell Eprimer32 what task to perform. Legal values are 1: Pick PCR primers, 2: Pick forward primer only, 3: Pick reverse primer only, 4: No primers needed. (Values: 1 (Pick PCR primers); 2 (Pick forward primer only); 3 (Pick reverse primer only); 4 (No primers needed)) -hybridprobe toggle [N] An internal oligo is intended to be used as a hybridization probe (hyb probe) to detect the PCR product after amplification. * -mishyblibraryfile infile Similar to MISPRIMING-LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library ...
Picks PCR primers and hybridization oligos Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall The sequence from which to choose primers. The sequence must be presented 5 to 3 [-outfile] outfile [*.eprimer3] Whitehead primer3_core program output file Additional (Optional) qualifiers (* if not always prompted): -[no]primer toggle [Y] Tell EPrimer3 to pick primer(s) * -task menu [1] Tell EPrimer3 what task to perform. Legal values are 1: Pick PCR primers, 2: Pick forward primer only, 3: Pick reverse primer only, 4: No primers needed. (Values: 1 (Pick PCR primers); 2 (Pick forward primer only); 3 (Pick reverse primer only); 4 (No primers needed)) -hybridprobe toggle [N] An internal oligo is intended to be used as a hybridization probe (hyb probe) to detect the PCR product after amplification. * -mishyblibraryfile infile Similar to MISPRIMING-LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather ...
Dear all, I obtained the job and need to identify presence of some bacteria in clinical samples by PCR with species/strain specific primers. The primers are already available in the lab, but the person who designed/find/order the primers were not available in the lab anymore and nobody knows the details. Therefore, I know primer sequences, the names of the strains, but I do not know the target (genome, 16S, ...), the size of the amplicon - actually I have no data to check the PCR result... I tried to blast them, but only one pair is found on the NCBI. How to find the target sequences, can anyone help me. Are there are softwares or databases for blasting the bacterial genome/transcriptome? ...
use Bio::PrimerDesigner; my $pd = Bio::PrimerDesigner-,new; # # Define the DNA sequence, etc. # my $dna = CGTGC...TTCGC; my $seqID = sequence 1; # # Define design parameters (native primer3 syntax) # my %params = ( PRIMER_NUM_RETURN =, 2, PRIMER_SEQUENCE_ID =, $seqID, SEQUENCE =, $dna, PRIMER_PRODUCT_SIZE =, 500-600 ); # # Or use input aliases # %param = ( num =, 2, id =, $seqID, seq =, $dna, sizerange =, 500-600 ); # # Design primers # my $results = $pd-,design( %params ) or die $pd-,error; # # Make sure the design was successful # if ( !$results-,left ) { die No primers found\n, $results-,raw_data; } # # Get results (single primer set) # my $left_primer = $results-,left; my $right_primer = $results-,right; my $left_tm = $results-,lefttm; # # Get results (multiple primer sets) # my @left_primers = $results-,left(1..3); my @right_primers = $results-,right(1..3); my @left_tms = $results-,lefttm(1..3 ...
Color Studio Professional most advanced primer ever. Created for skin repair and conditioning giving flawless results with any foundation.The DNA primer is a silky cream primer that contains a mix of anti-blemish and focused whitening agents that...
This is the amplicon context sequence in accordance with the minimum information for the publication of real-time quantitative PCR experiements (MIQE) guidelines. For more details, please refer to the following publication, Primer Sequence Disclosure: A Clarification of the MIQE Guidelines. ...
This is the amplicon context sequence in accordance with the minimum information for the publication of real-time quantitative PCR experiements (MIQE) guidelines. For more details, please refer to the following publication, Primer Sequence Disclosure: A Clarification of the MIQE Guidelines. ...
Usually the sequencing primer is used at 0.3µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.. For PyroMark Q24 and PyroMark Q96 MD the final concentration of the sequencing primer is 0.3µM and for PyroMark Q96 ID 0.4µM ...
Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of
There are three choices of RT primer: oligo(dT), random primers, or a gene-specific primer. The priming strategy has a strong impact on the workflow applied, since each priming method has prerequisites and consequences.. Oligo(dT) priming is used to receive full-length copies of the mRNA. For cDNA library construction or cDNA labeling applications, oligo(dT) primers are almost always used to prime cDNA synthesis. Anchored oligo(dT)18 primers are designed to bind at the beginning of the poly(A) tail (rather than randomly within the often long tail) to generate full-length cDNAs. This avoids mispriming and unnecessary reverse transcription of the long poly(A) tail. Since the 5´ ends of long mRNAs are often underrepresented in cDNA mixtures, this primer is preferred for most applications.. However, if the message is long or does not have a poly(A) tail (as with prokaryotic mRNA), then random hexamer primers are used. Random hexamer primers bind throughout the entire length of RNA, ensuring reverse ...
For each QuantiTect Primer Assay, we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design.. We then use our proprietary algorithm to design assays that amplify RNA sequences only (i.e., at least one primer overlaps a splice site), provided that information on the position of splice sites is available. The assays are designed to provide optimal performance with QuantiFast and QuantiTect SYBR Green Kits. After assay design, we validate randomly selected QuantiTect Primer Assays using real-time RT-PCR to check their compatibility with various real-time cyclers. Both two-step and one-step RT-PCR are carried out. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity (i.e., a single peak in melting curve analysis), and no primer-dimers in the no-template control (NTC). To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data ...
Cross-species primers have been used with moderate success to address a variety of questions concerning genome structure, evolution, and gene function. However, the factors affecting their success have never been adequately addressed, particularly with respect to producing a consistent method to achieve high throughput. Using 1,147 mammalian cross-species primer pairs (1089 not previously reported), we tested several factors to determine their influence on the probability that a given target will amplify in a given species under a single amplification condition. These factors included: number of mismatches between the two species (the index species) used to identify conserved regions to which the primers were designed, GC-content of the gene and amplified region, CpG dinucleotides in the primer region, degree of encoded protein conservation, length of the primers, and the degree of evolutionary distance between the target species and the two index species. The amplification success rate for the cross
Despite the many methodological options available for mutation detection, the choice is limited, by the necessity to ensure the highest sensitivity of detection for all types of changes while keeping costs and time of analysis within a reasonable limit. The present study extends to large and complex genes, like BRCA1, the applications of the FAMA method using large PCR amplicons (up to 1.4 kb). A key feature of this diagnostic strategy is the robust and economical two-step procedure for strand-specific labeling of PCR products using chimeric oligonucleotides and universal fluorescent primers (Fig. 1)⇓ . This labeling method is cost-effective, because it requires only one set of universal fluorescent primers, and allows reproducible amplification of all exons and splice sites of BRCA1 using homogeneous PCR conditions.. In the blind-test screening for changes in exon 11 (Table 2)⇓ different kinds of mutations and polymorphisms (various microdeletions/insertions involving 1 to 11 nucleotides as ...
Thermo Scientific™ M13/pUC sequencing primer (-20), 17-mer 10uM, 6nmol Unlabeled Oligonucleotides and Primers Oligonucleotides
Lasergenes PrimerSelect offers the best in advanced primer design software, enabling you to design and analyze primers for PCR, sequencing, probe hybridization and transcription.
Note: Optimal annealing temperatures are often higher than the Tm of the primers (approximately +5 to +10°C) and have to be determined empirically. For both primers, the Tm should be similar.. Bases that do not hybridize to the template may be added at the 5´ end of a primer (e.g., for introducing restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 µM are generally optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired product.. Note: For some systems, a higher primer concentration (up to 1 µM) may improve results. When testing new primers, always include a positive control reaction with a template that has been tested for function in PCR. This control shows whether the primers are working. The Human Genomic DNA from Roche is a good control template for evaluation of human primer ...
You need to first build an initial protocol and then optimize it.. The most important to determine the conditions is the polymerase used. Some polymerases work at higher or lower temperatures and will work faster or slower. Find the default protocol from the polymerase seller and this will give you a good idea where to start.. The second most important is the primers used. Depending on the GC % in the primers and their length, the times or temperature of each step will change, or if you prefer will need to be Optimized. The length of the DNA fragments will also determine the length of the cycles. Most polymerase sellers will includes steps to optimize the reaction depending on the quantity of DNA used, primer length and other parameters. For more information: Designing primers: Taq DNA polymerase protocol example:. Optimizing PCR ...
The first set of primers is designed to anneal to sequences upstream from the second set of primers, whereas the second set of primers is situated internally or nested with respect to the first set of primers. First set of primers also called outer primers amplify a large fragment of the gene which is used as a template in the second round of PCR that targets a smaller region of the amplicon using the second set of primers also known as inner primers or nested primers. The traditional approach to nested PCR was to perform a number of PCR cycles using first set of primers, and then open the reaction vessel and add the second, nested, set of primers to run second PCR cycle. The major problem with this approach is amplicon contamination in the laboratory and a consequential loss of specificity of the assay. To address this issue single-tube nested PCR (STNPCR) reactions have been developed, wherein both sets of primers are added to the initial reaction vessel and an extended PCR is performed. ...
Now more than ever, PCR has become a household name akin to Harry Potter or Gandalf the Grey, and in most cases no less mysterious nor magical. Whilst the distillation of molecular magic that is PCR has been invaluable (despite controversy) for screening your swabs, spit and other bodily fluids for COVID-19, it is also…
Further examination of the map shows that all of the reverse primers start at about the same place within the promoter region (~299,000), and the length differences are primarily due to choice of the forward primer, in the 3 downstream region of the gene. Since we are mostly interested in maximizing the promoter region, well choose Primer Pair 3 for further work. To save the sequence of the PCR product, along with its gene annotation, mouse over the map for Primer Pair 3 to bring up the menu shown above, and click on GenBank view. The GenBank view will appear in a new tab or window ...
Further examination of the map shows that all of the reverse primers start at about the same place within the promoter region (~299,000), and the length differences are primarily due to choice of the forward primer, in the 3 downstream region of the gene. Since we are mostly interested in maximizing the promoter region, well choose Primer Pair 3 for further work. To save the sequence of the PCR product, along with its gene annotation, mouse over the map for Primer Pair 3 to bring up the menu shown above, and click on GenBank view. The GenBank view will appear in a new tab or window ...
BiSearch software is composed of two basic algorithms. The first one is a primer design algorithm the second one is a search with the selected primers through genomic sequences to find potential non-specific PCR products.
BioMed Research International is a peer-reviewed, Open Access journal that publishes original research articles, review articles, and clinical studies covering a wide range of subjects in life sciences and medicine. The journal is divided into 55 subject-specific sections.
Chlamydiae are implicated in a variety of clinically and economically important diseases in livestock and companion animals. These bacteria are associated with abortion, conjunctivitis, encephalomyelitis, enteritis, pneumonia, and polyarthritis in ruminants. Infection with these bacteria is the most common cause of abortion in sheep and goats and also causes zoonotic infection in humans which, in pregnant women, can result in spontaneous abortion.
The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based
RAPD Primer Design from Metagenomes RPDM scans metagenome sequences identifying and determining relative frequency of candidate primers for Random Amplification of Polymorphic DNA RAPD assays
Applicability of the polymerase chain reaction (PCR) with species-specific primers to obtaining molecular markers for identification of the sibling species of Chironomus plumosus group - C. plumosus and C. balatonicus - has been estimated. The nucleotide sequences of internal transcribed spacer (ITS) from the locus encoding ribosomal RNA (rRNA) were used as the source for designing the species-specific primers. The primers allowing for identification of C. plumosus and C. balatonicus were constructed. One primer pair (plu107F/plu363R) gives the PCR product MAR2, specific of C. plumosus, and the other (bal86F/plu363R), the PCR product MAR6, specific of C. balatonicus. The testing involving 18 species of the genus Chironomus confirmed the specificity of the primers. The results suggest that the PCR with species-specific primers is promising for construction of molecular markers for identification not only of these two, but also of other Chironomus species.. doi: 10.5324/fn.v31i0.1381.. Published ...
Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a four- to eight-hour growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 minutes) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads
The Random Primer DNA Labeling Kit, Version 2 is designed for radioactive labeling for hybridization probes. Probe labeling can label DNA with [32P]-alpha-, [35S]-alpha- or [3H]-alpha-dCTP. This random primer DNA labeling kit is based on a modified method by Feinberg and Vogelstein and utilizes random oligonucleotide primers and cloned exonuclease-free E.coli DNA polymerase I, Klenow fragment. Using primers that are 9-mer and longer and exonuclease-free enzyme results in higher labeling efficiency and longer probes. This probe labeling method overcomes many of the disadvantages of conventional nick translation procedures while producing hybridization probes from very small amounts of DNA (10 to 20 ng). The Radioactive Labeling for Hybridization Probes kit can also be used to label DNA fragments that are embedded in low-melting temperature agarose gel slices.. ...
Two different molecular approaches, a multiplex PCR and PCR-RFLP of ITS-rDNA, were developed for the identification of Hoplolaimus species. DNA sequences of H. columbus, H. galeatus, H. concaudajuvencus, H. magnistylus, H. seinhorsti and three undescribed Hoplolaimus species were used to design species-specific primers. Three reverse species-specific PCR primers for H. columbus, H. galeatus and H. magnistylus were developed using the ITS1 region exhibiting interspecific variation. Three species-specific PCR primers in combination with the forward primer, Hoc-1f, produced distinct amplicons of 580 bp for H. columbus, 120 bp for H. galeatus and 340 bp for H. magnistylus. We successfully identified each of three species by multiplex PCR when all three were mixed in a single PCR reaction. Restriction enzyme digests of the PCR amplicon using HaeIII and RsaI permitted discrimination of H. columbus, H. galeatus, H. magnistylus, H. concaudajuvencus, H. sp. 1, H. sp. 2 and H. sp. 3 from each other. These results
Researchers showed how an isothermal amplification technique could detect S. enterica serovars from culture by targeting specific gene markers.
GenemerTM Products. The GenemerTM product line is PCR based. The product includes a specific primer pair for gene or mutation specific amplification. Genemer products are available for the gene fragment and disorders listed. Specialized optimized conditions may be required for certain triple repeat disorder amplifications. The GenemerTM kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. The product includes a specific primer pair for gene or mutation specific amplification, optimized buffers and dNTPs and in most cases, control DNA. These kits contain specialized and optimized conditions that are required for amplification of large repeats in certain triple repeat disorder amplifications. Gene Link recommends these GenemerTM kits for researchers who have not established their own optimized amplification conditions. GenemerTM kits are also available for conventional radioactive-based detection methods. A Radioactive component is not present in these kits. Gene Link ...
17138DNAArtificial SequenceSequence source PCR primer, 38 bases 1catgcatggg atccaacttg ctagacgcaa atatcgca 38239DNAArtificial SequenceSequence source PCR primer, 39 bases 2catgcatgcc cgggtcatga ttcttctttg atcatcacg 39328DNAArtificial SequenceSequence source PCR primer, 28 bases 3caccttctct tacgaatgtt tcgttggc 28439DNAArtificial SequenceSequence source PCR primer, 39 bases 4catgcatgcc cgggtcatga ttcttctttg atcatcacg 39539DNAArtificial SequenceSequence source PCR primer, 39 bases 5catgcatggg atccaactta ctggataaag aaagccgtt 39624DNAArtificial SequenceSequence source PCR primer, 24 bases 6tcaggcttct tcaatacaga ttgc 24737DNAArtificial SequenceSequence source PCR primer, 37 bases 7catgcatggg atccttcagc ttcgaactca gtaccga 37824DNAArtificial SequenceSequence source PCR primer, 24 bases 8tcaggcttct tcaatacaga ttgc 24928DNAArtificial SequenceSequence source PCR primer, 28 bases 9caccaacttg ctagacgcaa atatcgca 281024DNAArtificial SequenceSequence source PCR primer, 24 bases 10tcaggcttct tcaatacaga ttgc ...
The RT² qPCR Primer Assay is the most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis. Our experimentally verified design algorithm yields gene-specific qPCR assays characterized by uniform and high PCR efficiencies and standardized amplification conditions. Every RT² Primer Assay is subjected to rigorous experimental verification. Single product amplification of the correct size and high PCR efficiency are guaranteed when using the appropriate RT² qPCR Master Mixes. The uniform PCR amplification efficiencies and PCR conditions of the RT² qPCR Primer Assays provide an accurate and scalable solution for multiple gene expression analyses. Browse Primer Assays By Gene ...
The RT² qPCR Primer Assay is the most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis. Our experimentally verified design algorithm yields gene-specific qPCR assays characterized by uniform and high PCR efficiencies and standardized amplification conditions. Every RT² Primer Assay is subjected to rigorous experimental verification. Single product amplification of the correct size and high PCR efficiency are guaranteed when using the appropriate RT² qPCR Master Mixes. The uniform PCR amplification efficiencies and PCR conditions of the RT² qPCR Primer Assays provide an accurate and scalable solution for multiple gene expression analyses. Browse Primer Assays By Gene ...
Biological RNA generally comprises secondary structure motifs which cause a problem for target RNA detection by isothermal amplification methods. The complexity of the secondary structures makes RNA targets inaccessible for probe hybridization, resulting in decreased sensitivity and selectivity. This is particularly important because the hybridization step of the isothermal amplification method re ...