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:9/29/2014)... ZIONA, Israel , September 29, 2014 ... company developing novel immunomodulators for severe infections, today announced ... BARDA ) has awarded Atox Bio with a contract ... the development of AB103 to treat Necrotizing Soft Tissue ... public health threats that end in sepsis. ...
(Date:9/29/2014)... September 29, 2014 Florais de Saint ... its natural and soul-enhancing floral essences to the United ... essences into special formulas to help with specific treatments ... physical, mental, and emotional health. , “This is truly ... discoveries being made every day on how to properly ...
(Date:9/27/2014)... special section on single-photon detection, generation, and applications ... technologies with applications in areas as diverse as ... by providing more accurate measurement methods and new ... SPIE, the international society for optics and photonics, ... , The section appears in the August issue, ...
(Date:9/26/2014)... 2014 Synedgen announced today the ... (SBIR) Phase I contract from the National Cancer ... (NIH) to demonstrate proof-of-concept activity of Synedgen’s proprietary ... painful tissue damage to the lining of the ... this award, Synedgen will complete efficacy studies for ...
Breaking Biology Technology:Atox Bio Awarded Contract Worth up to $24 Million for the Development of AB103 by the Biomedical Advanced Research and Development Authority (BARDA) 2Florais de Saint Germain Brings New Kind of Flower Power To America 2Single-photon detection, generation, and applications featured in Optical Engineering 2Single-photon detection, generation, and applications featured in Optical Engineering 3Synedgen Awarded National Cancer Institute Funding for Promising New Treatment for Medical Radiation-induced Bowel Injury 2
... , , SAN FRANCISCO, Aug. 4 Nile ... the development of novel therapeutics for heart failure patients, today announced ... of its lead product, CD-NP, for the treatment of acute heart ... study is designed to provide additional information on the safety and ...
... /PRNewswire-FirstCall/ - Oncothyreon Inc. (Nasdaq: ONTY ) ... Hausman, M.D., as Vice President, Clinical Development at Oncothyreon. ... planning and implementing the clinical development program for the ... is an experienced oncologist who brings an extensive background ...
... , PALO ALTO, Calif., August ... antiviral therapies, announced today that it has licensed the exclusive ... Stanford University. This technology, discovered in the lab of Stanford ... focused on a variety of novel targets, including key features ...
Cached Biology Technology:Nile Therapeutics, Inc. Announces Dosing of First Patient in Phase 2 Study of CD-NP for the Treatment of Acute Heart Failure 2Nile Therapeutics, Inc. Announces Dosing of First Patient in Phase 2 Study of CD-NP for the Treatment of Acute Heart Failure 3Oncothyreon appoints Diana Hausman as Vice President of Clinical Development 2Oncothyreon appoints Diana Hausman as Vice President of Clinical Development 3Eiger BioPharmaceuticals Acquires Exclusive License to Novel Hepatitis C Virus (HCV) Technology From Stanford University 2
(Date:9/29/2014)... Energy Joint Genome Institute (DOE JGI), a DOE ... 32 new projects have been selected for the ... lakes to Caribbean waters, and from plant root ... in forested watersheds, the CSP 2015 projects portfolio ... be extracted. , "These projects catalyze JGI,s ...
(Date:9/29/2014)... who lived 2,330 years ago in the southernmost tip of ... when his DNA profile is one of the ,earliest diverged, ... region where modern humans are believed to have originated roughly ... ,mitochondrial DNA, was sequenced to provide clues to early modern ... that we all come from Africa, and helps us map ...
(Date:9/29/2014)... Ill. Scientists have discovered a previously unknown ... grow and, in the case of estrogen-positive breast ... work reveals new targets for breast cancer therapy ... the most aggressive treatment. , The University of ... Oncogene . , Estrogen pre-activates the unfolded-protein ...
Breaking Biology News(10 mins):2015 DOE JGI's science portfolio delves deeper into the Earth's data mine 22015 DOE JGI's science portfolio delves deeper into the Earth's data mine 32015 DOE JGI's science portfolio delves deeper into the Earth's data mine 42015 DOE JGI's science portfolio delves deeper into the Earth's data mine 52015 DOE JGI's science portfolio delves deeper into the Earth's data mine 62015 DOE JGI's science portfolio delves deeper into the Earth's data mine 72015 DOE JGI's science portfolio delves deeper into the Earth's data mine 82015 DOE JGI's science portfolio delves deeper into the Earth's data mine 9Ancient human genome from southern Africa throws light on our origins 2Ancient human genome from southern Africa throws light on our origins 3Scientists discover a new role for estrogen in the pathology of breast cancer 2
... Hopkins researchers have created a synthetic protein that, when activated ... body where cancer, arthritis and other serious medical disorders can ... type of diagnostic imaging technology and may someday serve as ... where signs of disease have been found. In a study ...
... mood for food can be changed by a restaurant,s choice ... calorie intake, according to a new study., "When we did ... music and lighting led diners to eat 175 fewer calories ... Wansink, professor of marketing and director of Cornell University,s Food ...
... FARMINGTON, Conn., Aug. 29, 2012 /PRNewswire-iReach/ -- The biobanking market ... new drug and treatment development for specific diseases. The ... and is projected to increase by 30% between 2010 and ... Private sector biobanks will experience the greatest increase with growth ...
Cached Biology News:Collagen-seeking synthetic protein could lead doctors to tumor locations 2Collagen-seeking synthetic protein could lead doctors to tumor locations 3Global Biobanking Market Projected to Increase by 30% to Reach $183 Billion by 2015 2Global Biobanking Market Projected to Increase by 30% to Reach $183 Billion by 2015 3
...
One-step, microplate or cuvet, colorimetric, detection range 0.3 g/dL to 5 g/dL. Procedure: 5 min....
Donor Goat Serum...
The Varian 4000 GC/MS system offers three easily switchable ionization configurations: internal ionization (II), external ionization (EI) or hybrid chemical ionization (CI)...
Biology Products: