Navigation Links
General Notes on Primer Design in PCR*

annealing efficiency: in general, the longer the primer, the more inefficient the annealing. With fewer templates primed at each step, this can result in a significant decrease in amplified product. The primers should not be too short, however, unless the application specifically calls for it. As discussed below, the goal should be to design a primer with an annealing temperature of at least 50C.

The relationship between annealing temperature and melting temperature is one of the Black Boxes of PCR. A general rule-of-thumb is to use an annealing temperature that is 5C lower than the melting temperature. Thus, when aiming for an annealing temperature of at least 50C, this corresponds to a primer with a calculated melting temperature(Tm) ~55C. Often, the annealing temperature determined in this fashion will not be optimal and empirical experiments will have to be performed to determine the optimal temperature. This is most easily accomplished using a gradient thermal cycler like Eppendorf's Mastercycler gradient.

Melting Temperature (Tm)

It is important to keep in mind that there are two primers added to a PCR reaction. Both of the oligonucleotide primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work at all since the primer with the higher Tm will mis-prime at lower temperatures and the primer with the lower Tm may not work at higher temperatures.

The melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula:

Tmprimer =
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. General Considerations for Successful Transfection Experiments
2. Real-Time PCR: General Considerations, Rev A
3. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
4. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
5. Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance
6. RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes
7. Gene-Specific Primers for NMDA Receptor Subunits
8. RT-PCR Primers for the Study of Apoptosis
9. New PCR Primers for Human Diseases
10. Amplification of Epstein-Barr Virus Exon C with Difficult Primers Using Eppendorf Mastercycler gradient
11. Human t-PA Control Primer Set
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: General Notes Primer Design PCR

(Date:7/25/2014)... MA -- Trillions of bacteria live in each person,s ... help digest food and stave off harmful infections, but ... , To help shed light on the role of ... associate professor Eric Alm recently tracked fluctuations in the ... year. The findings, described in the July 25 issue ...
(Date:7/25/2014)... (PRWEB) July 25, 2014 The Conferee ... for Topics for Conferee Networking. These two-hour sessions provide ... to meet and resolve problems, discuss new techniques, or ... deadline to submit a topic for consideration is August ... pittcon.org . The Conferee Networking committee will review the ...
(Date:7/25/2014)... Sinovac Biotech Ltd. (Nasdaq: SVA ), a leading ... today announced that it will release its unaudited financial results ... close on Thursday, August 14, 2014 EDT. The Company will ... Friday, August 15, 2014, at 8:00 a.m. EDT (Friday, August ... the Company,s financial results and provide an update on recent ...
(Date:7/24/2014)... , July 24, 2014  Asterias Biotherapeutics, Inc. ... emerging field of regenerative medicine, announced today that ... presentation to investors on Tuesday, July 29, 2014, ... presentation will include an overview of Asterias, business ... webcast, visit http://www.ustream.tv/channel/asterias-biotherapeutics at least 15 ...
Breaking Biology Technology:Monitoring the rise and fall of the microbiome 2Monitoring the rise and fall of the microbiome 3Pittcon 2015 Announces Call for Topics for Conferee Networking Sessions 2Sinovac to Host Conference Call to Report Second Quarter 2014 Unaudited Financial Results 2Asterias Biotherapeutics Announces Live Investor Webcast 2Asterias Biotherapeutics Announces Live Investor Webcast 3Asterias Biotherapeutics Announces Live Investor Webcast 4
... S.C., April 23 Signalife, Inc.,(Amex: SGN ... and Trademark,Office has awarded the Company a new ... Monitoring Heartbeats Of A Patient". The patent, which,covers ... develop new,proprietary products., Patent attorneys from Fulwider ...
... Novel Nucleoside Analogue; New ... CEO and Directors Appointed, ... announced today that on April 18, 2008 it acquired,Virium Pharmaceuticals ... with a portfolio of,clinical stage product candidates focused in oncology, ...
... Champions,Biotechnology, Inc. (OTC Bulletin Board: CSBR), a company ... tumor specific data to,enhance the value of oncology ... financing. Under the terms of the private investment,Champions ... stock at $1.75 per share. There were no ...
Cached Biology Technology:Signalife Awarded New U.S. Patent, 'System For, And Method Of, Monitoring Heartbeats Of A Patient' 2MacroChem Acquires Virium Pharmaceuticals 2MacroChem Acquires Virium Pharmaceuticals 3MacroChem Acquires Virium Pharmaceuticals 4MacroChem Acquires Virium Pharmaceuticals 5Champions Biotechnology, Inc. Completes $2,500,000 Private Financing at $1.75 per Share 2Champions Biotechnology, Inc. Completes $2,500,000 Private Financing at $1.75 per Share 3
(Date:7/25/2014)... (July 24, 2014)The link between autism and disrupted ... puzzle of the disease, and is largely unknown. ... Autism Research Initiative (SFARI), George Washington University (GW) ... offer truly integrative and in-depth answers to these ... , LaMantia, professor of pharmacology and physiology ...
(Date:7/25/2014)... from 25 countries and 35 states will attend ... Genetics Society of America (GSA) next week at ... will feature close to 500 presentations (including 70 ... gene expression and regulation, functional genomics, chemical biology ... a variety of diseases. , Of special ...
(Date:7/25/2014)... increasingly consume the healthcare community and remain ... , To address the worldwide crisis of ... countries, global leaders in diabetes, epidemiology and ... prevention and development of diverse intervention programs ... , This book on "Global Health Perspectives ...
Breaking Biology News(10 mins):GW researcher receives grant to answer the how and why of autism during development 2Yeast meeting to showcase latest breakthroughs in genetics and molecular biology 2What constitutes an effective response to the global 'diabesity' tsunami? 2
... available in French . , Researchers at McGill ... Jewish General Hospital along with colleagues at the University ... a significant breakthrough in the use of viruses to target ... Their results were published in the September early edition of ...
... Research today announced that Stuart A. Lipton, M.D., Ph.D., ... Aging, and Stem Cell Research Center at Burnham has ... a Center for Neurodegeneration Science (CNS). The center will ... the center comes from the National Institute of Environmental ...
... Innes Centre and the Central Science Laboratory could help ... stress and climate change. The research also shows that ... the climate. "While many compounds have been reported ... have identified how such compounds change in crops under ...
Cached Biology News:Viral 'magic bullet' targets cancer cells with help of new compound 2Burnham researcher awarded $8 million grant 2