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

endonuclease Sca I, followed by sequencing of all restriction-positive clones using the primer 5'-TGAATTTTCTGTATGAGG-3', the T7 Sequenase 2.0 kit, and 33PdATP from Amersham, according to the manufacturers instructions.

Results and Discussion

Although a T4 DNA ligase-overexpressing E. coli strain that allows cloning of linear DNA molecules was reported recently [4], efficient transformation of E. coli normally requires circular DNA. However, the formation of a circular structure in a three-fragment assembly ligation is a relatively rare event. At high DNA concentrations, which are crucial for a three-component assembly ligation [1], predominantly intermolecular ligation events leading to linear products occur, while DNA circularization is favored at low concentrations of the DNA fragments [5]. Our case was further complicated by the need to ligate two blunt-ended DNA fragments. Blunt-end ligation is known to be one to two orders of magnitude less efficient than cohesive-end ligation [6]. We chose to optimize the conventional procedure by sequential ligation of blunt- and cohesive DNA ends using different temperatures for the two processes. Therefore, we started the reaction at 26C, which was reported to be the optimal temperature for the joining of bluntended termini by T4 DNA ligase [7]. We followed this with cohesive-end ligation and circularization of our DNA at 14C, the temperature generally regarded as the best compromise to retain sufficient activity of the enzyme while still allowing relatively stable hydrogenbonded association of cohesive termini [8]. This strategy led to a more than 10-fold increase in the number of transformants obtained compared with a control ligation performed at single temperature (Table 1).

Using a protoco
'"/>

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:8/29/2014)... NY (PRWEB) August 29, 2014 Local ... Hills Veterinary Hospital are seeking candidates to participate in ... with osteoarthritis. The ultimate goal of this study ... stem cells into one or two arthritically affected joints ... joints. , Candidates for the current investigational study ...
(Date:8/29/2014)... A major challenge before new biotechnology start-up companies, ... Boston-Cambridge, is gaining visibility that can lead to important ... of Boston’s Adult Stem Cell Technology Center, LLC (ASCTC), ... his company an important priority since he started the ... social media marketing campaign launched earlier in ...
(Date:8/28/2014)... 2014 Best Sanitizers, Inc., a leader ... processing industry, is asking industry professionals to prepare for ... the E2 soap they’re currently using to the Best ... Hand hygiene is critical to fighting cross-contamination and ... Six key criteria are identified to evaluate the effectiveness ...
(Date:8/28/2014)... WEST LAFAYETTE, Ind. A new research ... "nanomechanical" properties of tiny structures undergoing stress ... insights to improve designs for microelectronics and ... spectroscopy, reveals information about how heating and ... their mechanical properties. Researchers have discussed the ...
Breaking Biology Technology:Burnt Hills Veterinary Hospital Seeks Candidates for an Investigational Study of Stem Cells for Dogs with Arthritis 2The Adult Stem Cell Technology Center, LLC Participates in Multiple Stem Cell and Regenerative Medicine Conferences Before Year End 2The Adult Stem Cell Technology Center, LLC Participates in Multiple Stem Cell and Regenerative Medicine Conferences Before Year End 3The Adult Stem Cell Technology Center, LLC Participates in Multiple Stem Cell and Regenerative Medicine Conferences Before Year End 4Best Sanitizers, Inc. Asks Food Industry Professionals: With Fall Harvest On the Way, Is Your E2 Hand Soap Up to the Task? 2New analytical technology reveals 'nanomechanical' surface traits 2New analytical technology reveals 'nanomechanical' surface traits 3
... Databases ... Receptors and Proteases, PARIS, August 18 Aureus ... life sciences discovery,announced today the release of three new ... for the important,drug targets Nuclear Receptor and Protease, as ...
... Aug. 18 LifeVantage Corporation,(OTC Bulletin Board: LFVN), the ... the progressive rate of aging, today announced,that the company,s ... interview on WallSt.net,s 3-Minute Press Show., The interview ... of the company,s latest press release., ...
... Inc. (OTC Bulletin Board: YGYB) ("Yongye" or the "Company"),a ... and,animals in the People,s Republic of China, today announced ... Second Quarter 2008 Highlights -- Revenue ... second quarter of 2007, and ...
Cached Biology Technology:Aureus Pharma Releases New Unified Knowledge Database 2[video] David Brown, President and CEO of LifeVantage Corporation, Discusses Record Page Views on WallSt.net's 3-Minute Press Show 2Yongye Biotechnology International Announces Second Quarter Results 2Yongye Biotechnology International Announces Second Quarter Results 3Yongye Biotechnology International Announces Second Quarter Results 4Yongye Biotechnology International Announces Second Quarter Results 5Yongye Biotechnology International Announces Second Quarter Results 6Yongye Biotechnology International Announces Second Quarter Results 7Yongye Biotechnology International Announces Second Quarter Results 8Yongye Biotechnology International Announces Second Quarter Results 9Yongye Biotechnology International Announces Second Quarter Results 10Yongye Biotechnology International Announces Second Quarter Results 11
(Date:9/2/2014)... and their propensity for eating anything, including each other, ... research from North Carolina State University finds that non-native ... eastern United States. , "The good news is that ... threat to humans," says Dr. Mary Jane Epps, a ... a paper about the research. , The research stems ...
(Date:9/2/2014)... Tithonian Konservat-Lagersttte of lithographic limestone is well known ... fossils from that area (for example, Archaeopteryx). Now, ... insects in the French equivalent of these outcrops ... the oldest known water treader. , Despite the ... fewer fossils have been obtained from the Late ...
(Date:9/2/2014)... DURHAM, N.C. -- When you accidentally touch a hot ... know the basic neural circuits involved in sensing and ... out the molecular players. , Duke researchers have made ... molecule involved in pain in worms, and have built ... Sept. 2 in Nature Communications , may help ...
Breaking Biology News(10 mins):Researchers find Asian camel crickets now common in US homes 2Exceptionally well preserved insect fossils from the Rhône Valley 2Surprising new role for calcium in sensing pain 2Surprising new role for calcium in sensing pain 3
... the Johns Hopkins School of Medicine and their colleagues have ... addition to their own can acquire new color vision, a ... new sensory information than anticipated. , This work, appearing ... ancestral primate inherited a new type of photoreceptor more than ...
... aren't as busy as, well, a bee. It all ... that a key to the insects' success in adapting to ... temperatures when flying to flowers. Whether and how they ... from the University of Washington and the University of Puget ...
... Schulich School of Medicine & Dentistry at The University ... Edinburgh, have discovered a way to kill the cystic ... of Dr. Miguel Valvano, Department of Microbiology and Immunology, ... of the Journal of Bacteriology, and highlighted in Nature ...
Cached Biology News:Making mice with enhanced color vision 2Making mice with enhanced color vision 3Bumblebee house warming -- it takes a village 2Bumblebee house warming -- it takes a village 3Key found to kill cystic fibrosis superbug 2
Rabbit polyclonal to Somatostatin 28 ( Abpromise for all tested applications). entrezGeneID: 6750 SwissProtID: P61278...
... Features Convenient platform for tube ... - 96 x 0.2ml V-bottom tubes ... Thermo-Tube Plates - Thermo-Fast® 96 Non-Skirted ... - 48 x 0.5ml Thermo-Tubes Multiple ...
... Pro upgrade for Shimadzu Phoretix 2D, Expression ... statistical tools for comparative gel analysis studies ... Data base add on component ... version 9i release 2 client software). ...
... systems provide linking and labelling reagents ... specific primary antibodies for visualising cellular ... accomplished using an ultra-sensitive indirect labelling ... technology. Secondary antibodies are polymerised directly ...
Biology Products: