Navigation Links
Optimizing the Direct Amplification of Missing cDNA 5' Ends,,, Using the Eppendorf Mastercycler gradient

an additional 5' area which completes the open reading frame. B. At the 5' end, the GC content of the isoamylase sequence is up to 80%, whereas the average GC content of the overall sequence is merely 52%. Discussion

With the aid of a simple PCR reaction, it was possible to amplify the 5' end of a cDNA coding for isoamylase directly from a ?ZAPII library. Conventional plaque screening of the same cDNA library produced several short isoamylase cDNAs, but no clone with a complete coding region. This finding may be attributable to the extremely high GC content of the 5' region of the isoamylase cDNA (Fig. 3b). In regions with high GC content, RNA is able to form stable secondary structures, which abort the cDNA synthesis and lead to shorter cDNA clones. Similarly, single-stranded DNA templates in a PCR reaction can form secondary structures that may interfere with amplification [3,4]. The higher the annealing temperature selected for the PCR reaction, the more likely secondary structures will be suppressed. This may explain why annealing temperatures of 69C or even 72C lead to more specific product than a temperature of 63C, although 63C is much closer to the theoretically optimal temperature of 61C. Determining a suitable stringent annealing temperature is crucial for obtaining specific products in the PCR. In this case, temperature gradients such as those on Mastercycler gradient enable systematic, time-saving experiments.

Bibliography

  1. Ohara, O., Dorit, R., and Gilbert, W. (1989) One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. USA 86,
    '"/>


Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
2. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
3. Optimizing electroporation parameters
4. Optimizing Büchi® Rotary Evaporator Applications
5. Optimizing electroporation parameters
6. Optimizing Büchi® Rotary Evaporator Applications
7. Optimizing Bchi Rotary Evaporator Applications
8. Optimizing electroporation parameters
9. Optimizing electroporation parameters
10. Optimizing electroporation parameters
11. Optimizing electroporation parameters
Post Your Comments:
(Date:8/26/2015)... and BEIJING , August 26, 2015 ... Impressive Acheivements  DiaCardio wins 1 st place  ... The competition, led in Israel by venture ... startups from countries including the US, China , ... Latin America .  Impressive achievement for ...
(Date:8/26/2015)... England , August 26, 2015 ... Ltd against Europlasma NV relating to P2i United ... have resolved their dispute in the United States ... liability on the part of either party. As a result ... Central District of California dismissed the ...
(Date:8/26/2015)... CA. (PRWEB) , ... August 26, 2015 , ... ... conferences to develop new business relationships with local sponsors in southern California. The Clinical ... facilitate their drug development processes and regulatory pathways., , ...
(Date:8/25/2015)... , Aug. 25, 2015   WuXi PharmaTech ... R&D capability and technology platform company serving the pharmaceutical, ... Ltd. ("Lee,s Pharm," Hong Kong Exchange Stock Code: 0950), ... Kong with 20 years of operations in ... two companies have signed an agreement whereby WuXi,s Laboratory ...
Breaking Biology Technology:Israeli Startup Takes First Place at Shengjing Global Innovation Awards 2015 2Israeli Startup Takes First Place at Shengjing Global Innovation Awards 2015 3Israeli Startup Takes First Place at Shengjing Global Innovation Awards 2015 4Israeli Startup Takes First Place at Shengjing Global Innovation Awards 2015 5Israeli Startup Takes First Place at Shengjing Global Innovation Awards 2015 6P2i Settles United States Litigation 2PRC Clinical To Attend Major Industry Conferences And Meet Sponsor Companies In The San Diego Biotech Hub 2PRC Clinical To Attend Major Industry Conferences And Meet Sponsor Companies In The San Diego Biotech Hub 3WuXi PharmaTech to Be Exclusive Supplier of Laboratory Testing Services to Lee's Pharm 2WuXi PharmaTech to Be Exclusive Supplier of Laboratory Testing Services to Lee's Pharm 3
... - The technological march has added so much complexity ... its promised benefits. , ,That according to John Swainson, ... delivered the opening keynote address at the 2007 ... to help bridge the relationship between business and information ...
... Wis. - The prospect of e-discovery and the possibility - ... any chief executive reach for antacids, but if the CEO's top ... of heartburn can be avoided. , ,That was the message of ... Best & Friedrich , who told a Fusion 2007 gathering that ...
... deal that will bring asset-tracking solutions to healthcare and ... a joint sales and marketing agreement with PanGo ... systems, technology, and services to buildings - including more ... the agreement will expand its wireless capabilities and provide ...
Cached Biology Technology:Fusion 2007: CA chief says IT complexity raises risk 2Fusion 2007: CA chief says IT complexity raises risk 3Fusion 2007: In e-legalities, CIOs and lawyers combine to provide business value 2Fusion 2007: In e-legalities, CIOs and lawyers combine to provide business value 3
(Date:8/12/2015)... YORK , Aug. 12, 2015  New ... hackers can attack Android phones to steal users, ... the increasing availability of fingerprint scanners on mobile ... utmost concern. To secure biometrics on mobile devices, ... to augment these convenient authentication systems with strong ...
(Date:8/6/2015)... Aug. 6, 2015  Today, U.S. Congressman ... Crossmatch ™ to learn firsthand how Silicon Valley ... Crossmatch is a world leader in multi-factor identity ... are helping federal agencies and commercial organizations achieve ... "The vast majority of network breaches ...
(Date:8/5/2015)... VIEW, Calif. , Aug. 5, 2015 ... exhibits continuous growth in applications, penetration into newer sectors, ... after year. The global biosensors space has seen the ... exited the market so far. (Photo - ... Frost & Sullivan, Analysis of the Global Biosensors ...
Breaking Biology News(10 mins):HYPR Corp. Addresses Android Vulnerability That Exposes Ecosystem-wide Biometric Security Challenges 2HYPR Corp. Addresses Android Vulnerability That Exposes Ecosystem-wide Biometric Security Challenges 3Rep. Honda to Visit Crossmatch in Redwood City 2Innovative Biosensors Incite Use in Non-Traditional Applications 2Innovative Biosensors Incite Use in Non-Traditional Applications 3Innovative Biosensors Incite Use in Non-Traditional Applications 4
... the president of Conservation International and a global leader ... Medal from the Harvard Museum of Natural History. ... who has written more than 500 scientific and popular ... has described 11 species new to science, had seven ...
... Scientists at UC Santa Barbara have made a significant discovery ... They explain nature,s way of controlling whether these cells will ... a liver, or any other part of the human body. ... the journal Cell . The scientists say the ...
... a successful second funding period, the first three DFG ... for another four years. This decision was made by ... Research Foundation) at its spring session in Bonn. As ... in the Earth System" in Bremen, "The Center for ...
Cached Biology News:Scientists shed light on inner workings of human embryonic stem cells 2Scientists shed light on inner workings of human embryonic stem cells 3The first DFG research centers to be funded for another four years 2The first DFG research centers to be funded for another four years 3The first DFG research centers to be funded for another four years 4
... is a 96-well ELISA-compatible plate (containing 12 x ... Monoclonal Antibody is immobilized by a method that ... the tail fiber protein of the T7 bacteriophage. ... 10 9 phage per well and low ...
... Telomerase Detection Kit is a ... for the fluorometric detection and ... activity. It incorporates refinements to ... adds the ability to quantitate ...
... The expertise that made the original Gene Pulser ... system available has been applied in the Gene ... The modular design of the Gene Pulser II ... accessory that has the widest range of settings ...
Caliper offers a wide array of standard LabChip products designed to be used in combination with our AMS 90 and LabChip 3000 microfluidic platforms....
Biology Products: