Navigation Links
Amplification Efficiency of TaqMan Gene Expression Assays

. Design parameters such as %GC content, Tm, and amplicon length were optimized to ensure that all of the TaqMan Gene Expression Assays have high amplification efficiency. Additionally, genome aided QC of all of the assays assures specificity for the target gene. This eliminates non-specific amplification a factor that could contribute to decreased amplification efficiency of the true target.

Testing Across a Broad Template Dilution Range

Total RNA from Stratagene Universal Human Reference RNA was reverse transcribed using random primers (Applied Biosystems High Capacity cDNA Archive kit) to generate cDNA. For each assay tested, 100 ng of cDNA was amplified with Applied Biosystems TaqMan Universal PCR Master Mix and the TaqMan Gene Expression Assay in a 50 μL volume for 40 cycles using universal cycling conditions (40 cycles of 95C for 15 seconds; 60C for 1 minute) on an ABI PRISM 7000 Sequence Detection System. This initial amplification was performed to produce sufficient template in the form of PCR product to enable the generation of a broad template dilution range.

A total of 5 μL of PCR product was run on a 4% agarose gel (NuSieve/ SeaKem) with size and quantitation standards (25 bp ladder from Invitrogen) to estimate the amount of product generated. The remaining 45 μL of PCR product was loaded on purification columns (QIAquick PCR purification kit; Qiagen) and purified according to the manufacturers directions. Bound DNA was eluted in a 100 μL volume of elution buffer. Then, 10 μL of the elute was used for picogreen staining (PicoGreen dsDNA quantitation kit, Molecular Probes) to estimate cDNA concentration.

A 6-log dilution range was generated using 10-fold serial dilutions of the target PCR product. Each of these dilutions was subjected to real-time PCR amplification as described above, using TaqMan'"/>

Source:


Page: All 1 2 3 4 5 6 7 8

Related biology technology :

1. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Greater Amplification Specificity with New Hot Start PCR Enzyme
4. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
5. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
6. Improve Amplification Specificity with Hot Start PCR Enzyme
7. Amplification of the amoA Gene of Nitrosococcus oceani Bacteria Using Eppendorf MasterTaq
8. Amplification of Mouse cDNAs for Microarrays Using the Eppendorf MasterTaq Kit
9. Optimizing the Direct Amplification of Missing cDNA 5 Ends Using the Eppendorf Mastercycler gradient
10. Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler
11. Amplification of Epstein-Barr Virus Exon C with Difficult Primers Using Eppendorf Mastercycler gradient
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Amplification Efficiency TaqMan Gene Expression Assays

(Date:7/29/2014)... --  Sequenom, Inc. (NASDAQ: SQNM ), ... solutions, and Mayo Medical Laboratories (MML), the ... the United States , have announced a license ... "We have great appreciation for Mayo Clinic,s commitment ... the opportunity to partner with the organization,s leading clinical ...
(Date:7/29/2014)... 2014) Tough, ultralight foam of atom-thick sheets can ... chemical process invented at Rice University. , In ... nanoscale building, with floors and walls that reinforce each ... materials: floors and walls of graphene oxide that self-assemble ... , The researchers say the foam could find use ...
(Date:7/29/2014)... NEW YORK , July 29, 2014  Pomerantz ... Impax Laboratories, Inc. ("Impax" or the "Company") (NASDAQ: IPXL).  ... Willoughby at rswilloughby@pomlaw.com or 888-476-6529, ext. ... certain of its officers and/or directors have violated Sections ... 1934.  On July 29, 2014, Impax ...
(Date:7/29/2014)... Pa. , July 29, 2014  Discovery ... dedicated to advancing a new standard of respiratory ... results on August 5, 2014 before the open ... The Company will host a live teleconference and ... During the conference call, Discovery Labs, management will ...
Breaking Biology Technology:Sequenom Enters Into License Agreement With Mayo Medical Laboratories 2Sequenom Enters Into License Agreement With Mayo Medical Laboratories 3Sequenom Enters Into License Agreement With Mayo Medical Laboratories 4Tough foam from tiny sheets 2SHAREHOLDER ALERT: Pomerantz Law Firm Investigates Claims On Behalf of Investors of Impax Laboratories, Inc. - IPXL 2Discovery Labs to Report 2014 Second Quarter Financial Results and Hold Conference Call on Tuesday, August 5, 2014 at 9:00 a.m. Eastern Time 2
... Christenson will assume duties as director of the state ... Secretary Cory Nettles announced the hire last ... initiative. , ,Part of the key to our ... in collaborative efforts. We hope to connect the tech ...
... For Ron Reynolds, an early peek at what would turn ... Way came through a hole in the ceiling. , ,As ... late 1970s, Reynolds sawed a hole in the ceiling of ... a specially built spectrometer through the roof of the building, ...
... of molten steel as the representative symbol of a ... the cubicle farm. Instead of coke and iron ore, ... data and information into knowledge and ideas: An invisible ... output is knowledge. Every physical product has a knowledge ...
Cached Biology Technology:Nettles Names Christenson as Director of Bureau of Entrepreneurship 2Journey Leads Astronomer to New Galactic Structure 2Journey Leads Astronomer to New Galactic Structure 3Transforming Information to Knowledge 2
(Date:7/29/2014)... Northern Territories in Canada and have sent smoke drifting ... on. NASA,s Aqua satellite collected this natural-color image ... July 26, 2014. Actively burning areas, detected by MODIS,s ... smoke are drifting northward in this image. Smoke ... Fire has caused power outages in the area and ...
(Date:7/29/2014)... Kent has shown for the first time how the ... could have a major impact on cancer therapy development. ... the mechanism whereby the physical properties of the internal ... are ,fine-tuned, to undertake different functions. , While some ... a framework for the cell, others are more dynamic, ...
(Date:7/29/2014)... ground breaking new book that brings together two of ... raise the profile of insect fossils through stunning photographs ... Penney and James E Jepson, details the incredible preservation ... setting the scene for what these remarkable fossils can ... even the future of our planet. Like the mosquito ...
Breaking Biology News(10 mins):Unique images bring fossil insects back to life 2
... engineered yeast strain can simultaneously consume two types of ... sugars are glucose, a six-carbon sugar that is relatively ... has been much more difficult to utilize in ethanol ... adding to earlier advances, reduces or eliminates several major ...
... 24, 2010) - Three weeks of oral resveratrol or ... microRNA activity close to their pre-event levels in excised ... were attributed to exerting the majority of the gene-switching ... microRNA optimally regulated by Longevinex and two optimally by ...
... New findings from the Monell Center reveal that weight ... formula the infant is consuming. The findings have implications related ... and other diseases later in life. "Events early in ... most significant influences is early growth rate," said study lead ...
Cached Biology News:Team overcomes major obstacles to cellulosic biofuel production 2Team overcomes major obstacles to cellulosic biofuel production 3Team overcomes major obstacles to cellulosic biofuel production 4Longevinex exerts greater influence over genes than resveratrol 2Longevinex exerts greater influence over genes than resveratrol 3Not all infant formulas are alike: Differential effects on weight gain 2
Mouse monoclonal [4i144] to HLA Class II DQ1 7 ( Abpromise for all tested applications). Antigen: Full length protein (Human): HLA Class 2 Antigen DQ1,7....
ASCIZ...
Goat polyclonal to CHMP5 ( Abpromise for all tested applications). Antigen: Synthetic peptide: C-NKDGVLVDEFGLPQ, corresponding to C terminal amino acids 202-215 of Human CHMP5 Entrez GeneID: ...
...
Biology Products: