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

>Isoamylase cDNA in ZAPII



Fig. 1 Diagram of the isoamylase cDNA in the ?ZAPII vector (Stratagene). The white rectangle represents the existing cDNA clone. The missing 5' region has a gray background. VP = vector primer (30-mer, Tm = 59C, 5'-GCT CAC TCA TTA GGC ACC CCA GGC TTT ACA), GP = gene-specific primer (30-mer, Tm = 66C, 5'-CAC CGG CTC GTC CTC CTC CCC CTC ATC CTC), S = isoamylase-specific probe. Method and results

The 5' end of a cDNA which encodes an isoamylase involved in starch metabolism, was isolated from wheat. A ?ZAPII cDNA library from wheat caryopses was used (approx. 2.5 x 107 pfu per reaction). In addition, the reaction mixture contained primers (0.3 mM each), nucleotides (0.2 mM each), 1.5 mM Mg2+ and Taq Polymerase (0.5 units). Aliquots (50 l) of a homogeneous reaction mix were used in a PCR-cycle with annealing temperatures ranging from 54C to 72C using the Mastercycler gradient. In order to identify specific products, the PCR products were separated (Fig. 2a) and hybridized against a DIG-labeled oligonucleotide probe of the 5' end of the original cDNA (Fig. 2b).

The annealing temperature for the PCR reaction described here was calculated to be 61C (PrimerSelect Software, DNA-Star Sequence Analysis Package). Both lower and much higher annealing temperatures were tested in order to find optimal reaction conditions. The size and complexity of the PCR product or mixture of products obtained was strongly dependant on the annealing temperature (Fig. 2a). A PCR blot analysis using an isoamylase-specific oligonucleotide pr
'"/>

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:4/21/2015)... SAN DIEGO , April 21, 2015 /PRNewswire/ ... instrument design and development, custom automation, and contract ... affordable and accessible flow cytometry and cell sorting ... collaboration to develop a novel flow cytometry platform. ... , NanoCellect Biomedical has developed a microchip-based ...
(Date:4/21/2015)... Calif. , April 21, 2015 /PRNewswire/ ... provider of advanced cryogenic logistics solutions for ... immunotherapies, stem cells, cell lines, clinical research ... announced that Dr. Mark W. Sawicki ... the 3rd Annual BioLogistics Summit in ...
(Date:4/21/2015)... , April 21, 2015  Novogen Limited (ASX:NRT; NASDAQ: ... company, advises that it has entered into definitive ... ordinary shares plus one attaching 6-month option and ... ordinary share issued, to institutional investors in ... private placement for aggregate gross proceeds of approximately ...
(Date:4/21/2015)... 2015 Progyny, Inc., the new combined ... that it has secured additional capital from investors including ... One and Merck Serono Ventures. The funding will enable ... best fertility outcomes by aligning science, technology, clinics and ... for families seeking fertility solutions and we are committed ...
Breaking Biology Technology:NanoCellect And Invetech Announce Collaboration To Develop Novel Flow Cytometry Platform For Cell Sorting And Analysis 2NanoCellect And Invetech Announce Collaboration To Develop Novel Flow Cytometry Platform For Cell Sorting And Analysis 3Cryoport to Present at the 3rd Annual BioLogistics Summit in San Francisco, CA 2Cryoport to Present at the 3rd Annual BioLogistics Summit in San Francisco, CA 3Novogen Conducts Private Placement and Announces Rights Offering to Shareholders 2Novogen Conducts Private Placement and Announces Rights Offering to Shareholders 3Novogen Conducts Private Placement and Announces Rights Offering to Shareholders 4Novogen Conducts Private Placement and Announces Rights Offering to Shareholders 5Novogen Conducts Private Placement and Announces Rights Offering to Shareholders 6Announcing Progyny: Combining Best-in-Class Clinical Solutions with Consumer Expertise 2
... 1, 2008 HighPoint Solutions,LLC, a premier provider of ... a global biotechnology company to design and implement,a Master ... new,product launches and support its expected rapid growth over ... strive to create a holistic view of,their customers in ...
... ARLINGTON, Va., April 1, 2008 Champions,Biotechnology, Inc. ... the,development of advanced preclinical platforms and tumor specific ... today the appointment of new,Board of Directors members ... it prepares to expand its operations., Abba ...
... Dr. Roger Kenneth,Hershline, PhD, MD, the CEO of Global ... charity CureHIV.us, announces today,that he is reaching out to ... Challenges in Global Health initiative. The,monies would be used ... treatment,method, LiSAVIOR., This unique new drug is also ...
Cached Biology Technology:HighPoint Solutions Selected by Global Biotechnology Company to Provide Master Data Management Solution 2Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 2Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 3Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 4Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 5Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 6Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 7Champions Biotechnology, Inc. Appoints New Board Members and Forms Management Team 8Cure HIV Reaches Out to Foundation Community to Fund LiSAVIOR 2
(Date:4/21/2015)... Canada , April 21, 2015 ... and evolving government policies are boosting access control systems ... According to a recently published report by ... Opportunities, 2020 ", the access control systems market in ... nearly USD1.2 billion by 2020.The access control systems market ...
(Date:4/20/2015)... -- Huntington Memorial Hospital is the first facility in ... wireless monitoring sensor to manage heart failure (HF). The ... heart failure monitoring device that has been proven to ... manage heart failure. The CardioMEMS HF System ... artery (PA) during a non-surgical procedure to directly measure ...
(Date:4/20/2015)... 20, 2015 The announcement comes ... Glenbeigh Records Management (GRM), Ireland,s foremost ... Bank. Having built up an impressive track record of clients ... player within the records management sector in Dubai ... is GCC staffbase and employ a further eight staff members ...
Breaking Biology News(10 mins):Saudi Arabia Access Control Systems Market to Touch USD1.2 Billion by 2020 Says TechSci Research 2Saudi Arabia Access Control Systems Market to Touch USD1.2 Billion by 2020 Says TechSci Research 3Huntington Memorial Hospital Becomes First in the San Gabriel Valley to Offer Patients a New Heart Failure Monitoring Solution 2Huntington Memorial Hospital Becomes First in the San Gabriel Valley to Offer Patients a New Heart Failure Monitoring Solution 3Glenbeigh Records Management Wins Contract With Dubai Islamic Bank 2Glenbeigh Records Management Wins Contract With Dubai Islamic Bank 3
... discovered the mother complete with her brood of some 20 ... of her soft part anatomy including legs and eyes. , ... of Leicester, Derek Siveter from Oxford, Mark Sutton from Imperial ... The team has made a digital image of the fossil ...
... Researchers at the National Institutes of Health have developed ... entire geographic regions, by eradicating the malaria parasite from ... tested only in mice, would prompt the immune system ... parasite from the digestive tract of a malaria-carrying mosquito, ...
... little mean sometimes ?especially when there's a choice bit of ... have shorter tempers than others. , Researchers in the ... of genes that affect aggression in the fruit fly Drosophila ... aggression in humans and other animals. , The study, ...
Cached Biology News:Malaria vaccine prompts victims' immune system to eliminate parasite from mosquitoes 2Malaria vaccine prompts victims' immune system to eliminate parasite from mosquitoes 3Fruit fly aggression studies have relevance to humans, animals 2Fruit fly aggression studies have relevance to humans, animals 3
6X Loading Buffer is intended for gel electrophoresis of DNA fragments or PCR products....
Ideally suited for resolving DNA and RNA 10-1000 bases especially PCR products ranging from 100-1000 bp. Gelling temp. (4%) 27C Melting temperature 65C. EEO: 0.10 Rnase/Dnase: none detected...
Agarose Gel Loading Dye Mixture 6X...
Rabbit Anti-JNK1/SAPK pan, Polyclonal Antibody...
Biology Products: