reaction

New Microarray Labeling Kit for Preparing cDNA Probes

...nverted into cDNAprobes in a reverse transcription reaction that incorporates labelednucleotides. The test and... and rbcL mRNA added to the probe-labeling reaction correspond to mRNA of high, medium, and low abundances, respectively. The relative fluoresce...

Assess the In Vivo Activation of Signal Transduction Pathways with PathDetect Reporting Systems

...re dishes. The total amount of DNA in each reaction was kept constant by adding quantities as needed of an unrelated plasmid (pBluescript plasmid). The pFC2-dbd plasmid, which expresses the GAL4 DNA binding domain but lacks an activation domain, was used as the negative contr...

Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library

... apo-B mRNA involves a site-specific deamination reaction that converts cytidine 6666 to uridine and generates a premature translational stop codon. 2 Although the catalytic subunit of the editing enzyme was discovered in 1993, the identity of the RNA-binding subunit has be...

Automated Fluorescent-Tag Cycle Sequencing

... ABI PRISM dye terminator cycle sequencingready reaction kit. The new Hot Top Assembly eliminates the need ...automatically determine thecorrect sequence of the reaction template with greater than 98% accuracy. 2 PE Applied Biosystems have found that, for their ABI PR...

Effects of Thermal Cycler Well-To-Well Temperature Uniformity on PCR Results

...ing patterns. When performing PCR amplifications, reaction components and PCR parametersare not the only crit...lowing thermal cycling, aliquots from each reaction were run on a vertical 6% polyacrylamide/TBE minigel, stained with ethidium bromide, and vis...

Brilliant Core Reagent Kits for QPCR and QRT-PCR

...uring several amplicons simultaneously in the same reaction vessel gives themost precise QPCR and QRT-PCR resu.... List Price U.S. List Price / reaction Catalog Brilliant QPCR SureStart Ta...

Genotyping Single Nucleotide Polymorphisms with Molecular Beacons

...n was monitored in real time while the PCR reaction was taking place. The fluorescent signal was monit...etermined to be above background while the reaction is in the exponential phase. Samples that showed no detectable increase in fluorescence thro...

Detecting Gastrin-Releasing Peptide Receptor by in situ PCR on Archived Tissue

...rse primer as used for reverse transcription. This reaction mixture isapplied to each tissue slice and covered... and developed with NBT/BCIP solution. The reaction is stopped by washing in PBS, followed by dehydration of the tissue in graded ethanols ( fig...

New Temperature Cycler Features Interchangeable Blocks

...ne preset hot blockto another, avoiding the longer reaction times associated with ramping the blocktemperature...le. The Hot Top Assembly heats thetop and sides of reaction tubes, significantly reducing condensation andeliminating the need for oil overlays on samples. 2,3 ...

New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences

...dy-to-use pESP-2 vector, PfuDNA polymerase and 10X reaction buffer, Epicurian Coli XL10-Goldultracompetent c...fusion protein, 20 g was cleaved in a 60-l reaction volume using Stratagenes Enterokinase (figure 3B) at room temperature for 10 or 20 hours. A...

Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood

...idual whole blood samples showed that each reaction produced the expected 107-bp PCR product and, therefore, the isolated genomic DNA is suitable for use in clinical reference laboratory assays (data not shown). Conclusions The quick and easy-to-use DNA Stat is the c...

Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase

...polymerase, longer extension times or modified PCR reaction buffers. 3 In general, however, PCR product yields...cessfully amplified the 0.9-kb target. The reaction products shown in lanes 1-7 were generated using 2 to 2.5 U of each DNA polymerase, with the...

Oil-Free PCR Using the Hot Top Assembly

...boCycler units fitted with Hot Top Assemblies heat reaction tubes from thetop and sides as well as the bottom....ssembly Unlike conventional PCR experiments where reaction volumes of 25 to 100 lare common, cycle sequencing experiments are performed using very small reacti...

Enhanced PCR Cloning System

...h PCR products are incubated for a 1-hour ligation reaction with one ofthe Srf I-digested PCR-Script vectors, Srf I restriction enzyme,and T4 DNA ligase. The Srf I enzyme recognizes the rare oligonucleotidesequence 5 -GCCCGGGC- 3 .By including the Srf I enzyme in the ligation reaction, the PCR-Scriptclonin...

Efficiently Insert Unique Restriction Sites into Plasmid Vectors

... plasmid miniprep kit 3 ), QuikChange 10X reaction buffer (5 l), primers (1 l of each at a co...he samples to cool slowly to room temperature. The reaction mixture was placed on ice while adding 1 l of PfuTurbo DNA polymerase (2.5 U), QuikC...

Pfu DNA Polymerase as a Hot Start Alternative

...high-fidelity DNA synthesis by thepolymerase chain reaction (PCR). Applications for Pfu DNA polymeraseinclude ... likely to occur during (1) preparation of the PCR reaction mixtures, (2) ramping to the initial denaturation temperature and (3) successive pri...

Comparing Fidelity and Performance of Proofreading PCR Enzymes

...ith respect to sensitivity. Using the standard PCR reaction conditions described above, PfuTurbo DNA polymerase synthesized a 4-kb product from as little as 10 fg of lambda DNA ( Figure 3 ). In contrast, at least 5 ng of lambda DNA was consistently required for amplifications c...

PCR Performance Comparisons Between pfuturbo and Taq DNA Polymerases

...xtension cycle of72C for 10 minutes. The amplified reaction products were electrophoresed on a1% agarose/1X TBE gel, stained with ethidium bromide, and imaged using the EagleEye II Still Video System. Lanes labeled M contain the kb DNAmarkers. REFERENCES Hogrefe, H. et al. (97) Strategies 10: 93-9...

prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

...th an appropriate thermostable DNA polymerase. The reaction tubemust be opened after cDNA synthesis. PCR reage...the RT and the DNA polymerase, under theparticular reaction conditions employed (e.g., number of duplications, pH, and[Mg 2+ ]). The mutation frequency (M.F.) o...

pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning

... amplicon was incubated at 72C for 30 minutes in1X reaction buffer, 100 M dNTPs, and 0.5 unit of Pfu DNApolyme...n 10% in these experiments if no polishing reaction was performed (data not shown). Conclusions PfuTurbo DNA polymerase is a high-performance, high-fi...

Production of Full-Length, Biologically Active MEK1WT and MEK1CA

...mCi of [ g - 32 P]ATP. Proteins were added to each reaction as indicated in figure 2 , and the reactions were incubated at 30C for 15 minutes and then mixed with 40 l of 2x SDS-PAGE sample buffer. REFERENCES Crews, C.M., Alessandrini, A., and Erickson, R.L. (1992) Science 258: 478-480. Zh...

Performance Comparisons of Commercial RT-PCR Systems

...e ProSTAR HF single-tube RT-PCR system,50-l RT-PCR reaction mixtures contained 1X HF RT-PCR buffer, 200 M each... ng/l oligo(dT), and RNA (200ng of total RNA). The reaction mixtures were incubated at 65C for 5 minutesand cooled to room temperature to allow the primers to a...

High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors

... XL kit. Electrophoresis of Dpn I-digested reaction products showed that bands of the appropriate size...on of XL10-Gold cells with a QuikChange XL reaction produced 32 colonies. In contrast, no colonies were produced with the standard QuikChange ki...

A New PCR-based Mycoplasma Detection Method

...PCR products from a standard amplification reaction were subjected to restriction analysis with Sau 3A and analyzed using agarose gel electrophoresis. The restriction patterns for each mycoplasma-derived PCR product were unique ( Figure 1 ); hence, individual species could now ...

Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal Performance

... of the DNA ends produced during the digestion reaction is compromised (i.e., a nucleotide is deleted), the lacZ gene is interrupted, and white colonies result. Enzymes tested in this assay (those commonly used in cloning applications) must produce less than 5% white colonies. Sta...

RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes

...ipts by the reverse transcriptase polymerase chain reaction (RT-PCR). TheRT-PCR primer sets for mismatch repai...ing RoboCycler temperature cyclers. Each reaction was performed in 1X Taq polymerase buffer, 250 M of each dNTP, 10 pmol of each primer and 2....

XL10-Gold Cells Supplied in New Single-Reaction Format

...wed and aliquottedinto Falcon 2059 polypropylene reaction tubes. To speed up thetransformation process, we designed the SoloPack format so that the entiretransformation reaction, including post heat-shock expression, is performed in asingle tube. The SoloPack single-tube, single-reaction format is available...

Greater Amplification Specificity with New Hot Start PCR Enzyme

...little or no modification of cycling parameters or reaction conditions. SureStart Taq DNA polymerase c...ctivity at temperatures encountered during reaction setup or while ramping up to stringent primer annealing temperatures. For example, Taq DNA p...

CastAway Precast Gels for Rapid Automated DNA Sequence Analysis

... Figure 2 DNA reaction products are heat denatured and loaded on the CastAway gel using a 48- or 64-well sharktooth comb. To separate DNA fragments produced in a sequencing reaction, electrophoresis is carried out at a constant 25 W (ca. 50 V/cm and...

Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations

...ygote) of two allelic variants in a single reaction tube ( Figure 1 ). Convenient Single-Tube Format Each assay kit includes two allele-specific molecular beacons with an exactsequence match to each of the two target sequence variants, genotype DNAcontrols (wild type/mutant homozygotes and h...

Easily Amplify Genomic DNA with Long-Distance PCR

...1 ). Following amplification, 5-l aliquots of each reaction mixture were analyzed by electrophoresis in 0.5X TBE 0.9% agarose gels, and the amplification products were visualized by staining with ethidium bromide. Table 1 Conditions Used toAmplify Mouse SMHC Gene Fragme...

Enzyme Immunoassay for Studying Intracellular Levels of cAMP

...d reagents are removed by a wash step. The reaction is completed by the addition of the colori...esults in a blue-green product background, and the reaction is stopped by the addition of 1 M phosphoric acid. A bright yellow color results, an...

Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini

...oteases, functions efficiently over awide range of reaction conditions and is provided with Soybean Trypsin In...leavage product and the free CBP tag (lane 2). The reaction was absorbed for 30 minutes with a mixed slurry of CaM affinity resin and STI Agarose ...

Using XL1-Red Mutator Strain to Generate Esterase Variants

...es (tedious and time consuming), alter the reaction conditions (e.g. change the solvent system ...hate buffer (50 mM, pH 7.5) at 40C.As expected, no reaction was observed either with the wild type (PFE) or in theabsence of enzyme. In contrast, by using varia...

Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence

...complementary single-stranded tails. The annealing reaction wastransformed into Epicurian Coli XL1-Blue superc...g of prepared pCAL-n-EK vector DNA. Onetenth ofthe reaction was transformed into XL1Blue supercompetent cells, resulting inapproximately 800 colony forming unit...

Constructing Directional cDNA Libraries from Limited Amounts of RNA

...oachthat combines linker-mediated polymerase chain reaction (PCR) for amplifyingcDNA and ligation-independent ... 100-l reactions in0.75-ml, thin-walled PCR tubes; reaction components were as specified in the kitsmanual. These reactions were amplified using the RoboCycler ...

Randomize Gene Sequences with New PCR Mutagenesis Kit

...mutation frequencies using a single set of optimal reaction conditions. The kit features Mutazyme ... the use of error-prone DNA polymerases and reaction conditions. 1,2 Randomized DNA sequences are cloned into expression vectors, and th...

Generate High-Quality, Directional Plasmid cDNA Libraries

...be limited by the efficiency of the cDNA synthesis reaction and the transformation of plasmid clones. Stratagene has improved the general methods for constructing plasmid cDNA libraries and offers three plasmid-based cDNA library construction kits using the pBluescript II (prokaryotic)...

Gene-Specific Primers for NMDA Receptor Subunits

... in reversetranscriptase-mediated polymerase chain reaction (RT-PCR) assays to showexpression of NMDA subunits with Stratagenes rat cDNA, human cDNA and hNTneuron cDNA libraries as templates. By using these primers, researchers will beable to study rat and human NMDAR subunit-specific expression in a variety ...

Unique DNA Polymerase Formulation Excels in a Broad Range of PCR Applications

...as asupplement rather than including it in the PCR reaction buffer ensures thatmaximal product yields are achieved. Yield and Target-Length Comparisons Fig.3 The PCR performance of Herculase enhanced DNA polymerase was tested relative to Platinum Taq DNA polymerase High Fidelity (...

Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase

...es perkb of target. 1 A complementary set of PCR reaction conditions has been developed to furtherimprove am...were observed when PCRs were performed in 1.5X PCR reaction buffer, compared to the 1X concentration ( Table 1 ). Table 1 PCR EnzymeError R...

High-Fidelity PCR with a Novel Polymerase Mixture

...Taq DNA polymerase in 1987, 1 thepolymerase chain reaction (PCR) amplification of DNA target regions has beco...ize PCR products up to 12 kb in cloned Pfu reaction buffer 13 and up to 25 kb in Taq reaction buffer ( table 1 ). ...

Highly Active and Removable AffinityTagged Lambda Phosphatase

...upernatant was assayed for phosphatase activity by reaction with thechromogenic substrate, p-nitrophenyl phosp... figure 2 After reaction of EGFRKD with Lambda Phosphatase and treatment with the CaM affinity resin to remove the ph...

HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA

...pulation of samples is required.Because the entire reaction takes place in one tube, the test saves time andeffort and significantly reduces the risk of PCR product carry-overcontamination. Furthermore, this multiplexed assay includes both an HSV-specificmolecular beacon as well as an internal control so assa...

Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer

...g temperature. For the long targets, the amplified reaction productswere electrophoresed on a 1% agarose/1X TBE gel. Difficult PCR Amplification Systems Figure 1 To demonstrate the variety of improvements Perfect Match PCR enhancer can have on PCR performance, we selected pr...

Identification of Differentially Expressed Gene Products with the CastAway System*

...fied by the RNAarbitrarily primed polymerase chain reaction (RAP-PCR). A human myelomonocytic cell line (HL60...rred to as RNA arbitrarily primed polymerase chain reaction (RAP-PCR) 1 and differential display (DD), 2 share several features. Bothtechniques rely on the deg...

Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance

...A targets (300 bp, 500 bp, 900 bp). Using the same reaction conditions, greater variation in amplicon yield was observed with Taq DNA polymerase (data not shown). PCR Fidelity Comparisons Herculase enhanced DNA polymerase exhibits high fidelity due to its novel pfu-based compos...

Tools for Detecting MSH2 Expression in Chinese Hamster Ovary Cells

...agments was labeled with a- 32 P-dCTPin a 5-minute reaction using Stratagenes Prime-It II random primer labelingkit. 8 The probes were purified to remove unincorporated nucleotidesusing Stratagenes NucTrap probe purification columns. 9 Thespecific activity of each probe was greater than 10 9 cpm/g. ...

RT-PCR Primers for the Study of Apoptosis

...y. Increasing the concentration of template in the reaction by 50% did not result in a significant increase of PCR products. Figure 3 The ICE-Lap-3 and Fas antigen primer sets did not amplify the expected PCRproduct from the human muscle cDNA library. Repeated PCR attempts ge...

Mammalian Expression Vectors with Interchangeable Selectable Markers

...ble hosts with the recombination reaction. Desired reaction products are obtained with greater than 90% effici...tic-resistance cassette, and Cre recombinase. This reaction is followed by heat inactivation of Cre recombinase at 65C for 15 to 30 minutes. The ina...

New Competent Cells for Highest Transformation Efficiencies

...for the ligation-independent cloning (LIC) reaction using standard conditions. 2 The kanamycin-resist...thesis Kit. The ligations were performed in a 15-l reaction volume using 30 ng of vector and 10 ng of library cDNA. Of this ligation reaction, 1...

Improve Amplification Specificity with Hot Start PCR Enzyme

...little or no modification of cycling parameters or reaction conditions. SureStart Taq can be used in a variety...merase activity at temperatures encountered during reaction set-up (4 to 25C) or while ramping up to stringent primer annealing temperatures. For example, Taq D...

Mx4000 Multiplex Quantitative PCR System*,PPP

...ct accumulation duringthe exponential phase of the reaction using fluorescence data and providesaccurate information on initial starting copy number. PCR amplification anddetection are combined in a single step, speeding the process of gatheringexperimental data. The QPCR process uses a single closed tube, wh...

Transgenic Animals

... identified clones which showed the desired reaction are multiplied in vitro and injected into blastocysts. These injected blastocysts are then implanted into the uterus of pseudo-pregnant females. ...

Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit

...dded to 15 l of the DNA sample for a total reaction volume of 25 l. The master mix was pipetted into the lid of the corresponding reaction tube. The lids were closed, and the tubes were briefly centrifuged to add the master mix to ...

Eppendorf Perfectprep Gel Cleanup Kit

... with 20 units of EcoRV in a 30 l reaction. The reaction was stopped by adding 3 l of 10x Gel Loadi...linearized with 40 units of SmaI in a 40 l reaction for two hours at 25C. Gel Electrophoresis: The entire reaction volume (30 l) of each...

An Assessment of the Eppendorf twin.tec plate performance in PCR* applications

...er commercial microplate supplier and compared the reaction products by agarose gel analysis. Addition... efficient transfer of block temperature to the reaction samplethis feature translates into a robust and reliable PCR reaction. Another desirable ...

A Unique Dimension in Biological Purity

... consumables. The absorbed EtO and its reaction products are not easily removed from these article... The levels of residues of EtO and its reaction product ethylene chlorohydrin that can be tolerated have to be very low, but even the sm...

Chemical Stability of Disposables

... These include: The chemical reaction with the polymer, e.g. by oxidation, reaction with the functional group, and by depolymerization Dissolving the synthetic mater...

TripleMaster PCR System

...fer choice Choosing the appropriate reaction buffer is essential for obtaining optimal re.... The resultant higher ionic strength of the reaction medium forces the TripleMaster Polymerase Mix to amplify short DNA fragments with a higher yie...