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:8/20/2014)... The 6th Annual Arrowhead Personalized and ... 29-30 in San Francisco, CA. This year’s conference brings ... and clinicians focused on addressing and solving the significant ... twelve years since the sequencing of the human genome, ... pharmacogenetic information in the label and molecular diagnostics have ...
(Date:8/20/2014)... Calif. , Aug. 20, 2014 /PRNewswire-iReach/ -- ... the transformative collaboration between the school,s bioengineering department ... part of the Intel® Software Academic Program, UCSD,s ... sciences and the human body.  ... The study exhibits the work of Dr. ...
(Date:8/20/2014)... 20, 2014 WriteResult®, a leading provider ... it has expanded its myPROpad™ ePRO tablet with the ... one of the most widely used and well-respected Patient ... status. The EQ-5D is used during clinical trials and ... that can be used to demonstrate Health-Related Quality of ...
(Date:8/19/2014)... One of the major limitations in ... vascular supply. Nutrient and gas transport throughout synthetic ... for successful regeneration and integration into the patient’s ... vascularization and promote angiogenesis in a variety of ... and architecture, and enhancing scaffolds with stem cells. ...
Breaking Biology Technology:6th Annual Personalized and Precision Medicine Conference Bringing Together Thought Leaders to Discuss Clinical Implementation, Dx Reimbursement, Big Data, and Sequencing 2Intel and University of San Diego Bioengineering Department Release Case Study on Health Sciences Research 2EQ-5D™ Assessment Now Available for iPad 2Vascularization in Tissue Engineering Highlighted at 12th New Jersey Symposium on Biomaterials Science 2
... The Defense Advanced Research Projects Agency (DARPA) has awarded ... Science an $8.4 million grant for research on a ... electronic devices to keep their state even while powered ... The research has broad implications across ...
... Physicians at Thomas Jefferson University Hospital reported today on ... automated interpretation of coronary CT angiography (cCTA) by Rcadia,s ... disease (CAD) in the major coronary arteries as well ... at the Radiological Society of North America (RSNA) Annual ...
... Dec. 2, 2010 The Board of Directors of ... capital investments and customized company formation and business growth ... the approval of the nomination of Peter M. DeComo ... PLSG, effective immediately.  Mr. DeComo will replace Dr. D. ...
Cached Biology Technology:UCLA receives $8.4 million to lead research on ultra-low-power, non-volatile logic technologies 2Rcadia COR Analyzer System Safely Rules Out Coronary Artery Disease in Main Coronary Arteries and Branch Vessels 2The Pittsburgh Life Sciences Greenhouse Announces New Chairman of the Board 2
(Date:8/20/2014)... the University of Tbingen, Arizona State University, the Wellcome ... Health Institute (Swiss TPH) isolated Mycobacterium pinnipedii ... 1000 years old. The pathogen is a relative of ... causes disease in humans today. These researchers assume that ... coast. "The link to sea lions was unexpected" comments ...
(Date:8/20/2014)... the Oklahoma Medical Research Foundation a five-year, $14.5 million ... bacteria,s effects on humans. , For 10 years, ... studied the human immune response to anthrax bacteria as ... original funding came soon after anthrax-laced letters killed five ... attacks of Sept. 11, 2001. , The long-term ...
(Date:8/20/2014)... spiciness that is irresistible to some, but intolerable to ... their findings to develop a new drug candidate for ... inflammation or other problems. They reported their progress on ... in ACS, Journal of Medicinal Chemistry . , ... had pegged a compound called capsaicin as the active ...
Breaking Biology News(10 mins):$14.5 million grant awarded to continue anthrax studies 2
... ANN ARBOR, Mich.---The muscle-building abilities of hormones known as ... search and you,ll find not only scientific papers discussing the ... muscle tissue, but also scores of ads touting the purported ... spite of widespread interest in these potent molecules, key details ...
... 2010) -- The popular forage and turf grass called tall fescue ... area that,s estimated to be larger than Virginia and Maryland combined ... Indiana University suggests there is more to fescue than meets the ... in the Journal of Applied Ecology , show that a ...
... ( P. vivax ) malaria has been identified in ... those who do not express the Duffy blood group protein ... Western Reserve University School of Medicine, Pasteur Institute, and the ... 600 individuals from eight communities covering the main malaria transmission ...
Cached Biology News:U-M researchers solve a molecular mystery in muscle 2Study: Grass, fungus combination affects ecology 2Duffy-negative blood types no longer protected from P. Vivax malaria 2Duffy-negative blood types no longer protected from P. Vivax malaria 3
HistoScan AP Universal Goat Kit...
Interact with both DNA and RNA; fluorescence green (Em=525 nm) bound to DNA; and fluorescence red (Em=~650 nm) bound to RNA....
One bottle containing 30 ml of protein solution and 0.1% sodium azide....
...
Biology Products: