Navigation Links
Cloning Based on Efficient Three-Fragment Assembly,,,DNA Ligation

Abstract

The efficiency of the Rapid DNA Ligation Kit from Roche Molecular Biochemicals was compared with standard and optimized conventional ligation procedures. The reactions generated circular recombinant bacteriophage DNA in a three-fragment assembly DNA ligation. Sequential blunt-end and cohesive-end ligation, including a temperature shift from 26C to 14C, led to a more than 10-fold increase in the number of transformants obtained. Applying a modified protocol, the kit was found to provide a reliable, fast, and remarkably efficient procedure for this complex application.

Introduction

To construct a phage displaying integrin-binding ligands, we needed a reliable and efficient ligation procedure, by which three DNA fragments could be assembled simultaneously, yielding circular DNA. Such a ligation is known to require high concentrations of the reacting DNA molecules [1], especially if blunt-ended DNA is involved [2] and is therefore usually circumvented by subcloning. Because of the absence of appropriate restriction sites in the DNA fragments, subcloning into another suitable vector and subsequent ligation of the two fragments in this construct was impossible in our case. A standard ligation protocol led only to poor transformation results, whereas parallel ligations using either an optimized conventional procedure or the Rapid DNA Ligation Kit from Roche Molecular Biochemicals resulted in efficient and correct formation of the desired cloning vector.

Materials And Methods

The bacteriophage fd-tet which confers tetracycline resistance upon infected cells was donate
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
2. Enhanced PCR Cloning System
3. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
4. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
5. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
6. QIAGEN PCR CloningPlus Kit
7. Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments
8. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
9. Development of a Multiplex Bead-Based Assay for Antibody Screening of a Nonhuman Primate Colony on the Bio-Plex System
10. A Mix-and-Read Cell-Based Assay for Hybridoma Screening Using the FMAT 8100 HTS System
11. Mix-and-Read, Bead- and Cell-Based Immunoassays Using the FMAT 8100 HTS System
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Cloning Based Efficient Three Fragment Assembly DNA Ligation

(Date:5/27/2015)... 27, 2015 Expanding in a big ... Avalon Biomed Inc. recently inked a deal ... Dental Co. Ltd. , the largest endodontic distributor in ... international market for Avalon Biomed, which manufactures Grey MTA ... silicate-based dental cements that have won renown from dentists ...
(Date:5/27/2015)... , May 27, 2015  Tikcro Technologies Ltd. (OTCQB: ... March 31, 2015. Aviv Boim ... continued pre-clinical work to generate functional specific antibodies, targeting ... stage, we are encouraged by the development progress. We ... new antibodies which modulate immune checkpoints gain clinical acceptance ...
(Date:5/27/2015)... Empowering biomedical researchers with richer, more ... today announced its MR Compatible Small Animal PET ... uses silicon photomultiplier detector technology, so it can ... manufacturers like Bruker and Agilent,” said James Schellenberg, ... enables simultaneous PET and MRI data acquisition with ...
(Date:5/27/2015)... , May 27, 2015  Northwest Biotherapeutics, Inc. ... developing DCVax® personalized immune therapies for cancer, announced today ... Officer of the Company, will be making an Industry ... in Chicago on Saturday, May ... the Industry Expert Theater in McCormick Place. ...
Breaking Biology Technology:Dental Manufacturer Avalon Biomed Inks Major Deal with Japanese Distributor 2Dental Manufacturer Avalon Biomed Inks Major Deal with Japanese Distributor 3Tikcro Technologies Reports First Quarter 2015 Results 2Tikcro Technologies Reports First Quarter 2015 Results 3Cubresa Launches Simultaneous PET/MRI Technology 2NW Bio To Present Overview Of DCVax-Direct Phase I Trial In Chicago This Weekend 2NW Bio To Present Overview Of DCVax-Direct Phase I Trial In Chicago This Weekend 3
... Calif., June 5 /PRNewswire-FirstCall/ - BioMarin Pharmaceutical,Inc. (Nasdaq ... its third,enzyme replacement therapy (ERT) for the treatment ... BioMarin plans to initiate a Phase,1/2 clinical trial ... two MPS drugs on the market, we plan ...
... ) notes that FDA has published a notice of a ... to,be held on July 23, 2008. The Committee will address ... " ... presentations from the Office of Generic Drugs (OGD) ... treat gastrointestinal (GI) conditions ... " For over ...
... Will be Presented at the American Society for Mass ... 56th ASMS Conference in Denver, June ... Inc.,(Nasdaq: BNVI ) today announced that it has ... platform that allows for the rapid, specific, sensitive,automated quantification ...
Cached Biology Technology:BioMarin Announces Program for ERT for Treatment of MPS IVA - Morquio A Syndrome 2BioMarin Announces Program for ERT for Treatment of MPS IVA - Morquio A Syndrome 3BioMarin Announces Program for ERT for Treatment of MPS IVA - Morquio A Syndrome 4Viropharma Comments On Upcoming FDA Advisory Committee Meeting 2Viropharma Comments On Upcoming FDA Advisory Committee Meeting 3Bionovo Develops a High Throughput LC-MS/MS Method for Simultaneous Quantification of Multiple Bioactive Compounds 2Bionovo Develops a High Throughput LC-MS/MS Method for Simultaneous Quantification of Multiple Bioactive Compounds 3
(Date:5/19/2015)... 2015 Research and ... of the  "Genetic Testing Market Outlook 2018" ... , ,A recent report, Genetic Testing Market ... the current and future genetic testing market. ... working principles and types are covered in ...
(Date:5/11/2015)... 11, 2015  Through a well-rounded UAS delegation representing private ... a strong showing at AUVSI,s Unmanned 2015 conference last week ... Ohio,s UAS industry met with over 200 ... points along the UAS ecosystem. "Our message ... President for Aerospace Rich Knoll . "If you want ...
(Date:5/6/2015)... TEL AVIV, Israel , May 6, 2015 /PRNewswire/ ... the world,s most established helmet producer, announced today that ... LifeBEAM Smart Helmet, the world,s first bio-sensing cycling helmet ... will be released in two new colors in order ... demand for the product. In addition, LifeBEAM and Lazer ...
Breaking Biology News(10 mins):Global Genetic Testing Market Outlook 2018 2Ohio Flies High at Unmanned '15, Sets Stage for Ohio UAS Conference 2LifeBEAM and Lazer Announce Expanded Partnership in Bio-Sensing Sports Gear 2LifeBEAM and Lazer Announce Expanded Partnership in Bio-Sensing Sports Gear 3
... Stanley, Inc. (NYSE:,SXE), a leading provider of systems ... announced today that it has entered into a,definitive ... intelligence operations and information technology services,company. The purchase ... to certain working capital and other adjustments as ...
... Calif.) Michael Gazzaniga, professor of psychology at UC ... the Study of the Mind, has received the Humboldt ... "Professor Gazzaniga is a true pioneer in reaching ... the relationship between mind and brain," said UCSB Chancellor ...
... Dozens of instruments to be deployed on the Oak ... provide valuable information related to climate change, biodiversity and ... Walker Branch Watershed and other portions of the ... National Ecological Observatory Network, a multi-decade continental-scale research platform ...
Cached Biology News:Stanley to Acquire Oberon Associates 2Stanley to Acquire Oberon Associates 3Stanley to Acquire Oberon Associates 4Neuroscientist Michael Gazzaniga awarded Humboldt Prize 2Oak Ridge pegged for national ecological network 2
Mouse polyclonal antibody raised against a partial recombinant QPCT. NCBI Entrez Gene ID = 25797...
... recombinant IFNA2. Immunogen: Human IFNA2 ... in E. coli. The biological activity was ... OMIM: 147562, GeneID: ... MA Code: ABV0P9DPB ...
... longer need to perform time consuming and inaccurate ... Surveyor, any found variants of the sample when ... in our mutation electropherogram as a sharp peak. ... analysis mode is over 99%, with sensitivity to ...
...
Biology Products: