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:5/29/2015)... , May 28, 2015 Research ... addition of Jain PharmaBiotech,s new report "Cytogenetics ... offering. This report deals with cytogenetics ... use mainly to describe the chromosome structure and ... of molecular biology, it is also referred to ...
(Date:5/28/2015)... , May 28, 2015 ... announced the addition of Jain PharmaBiotech,s new ... and Companies"  to their offering.  ... remains a challenge in management. With nearly ... the seven major markets of the world ...
(Date:5/28/2015)... -- Research and Markets( http://www.researchandmarkets.com/research/l9dqwt/animal_genetics ) has announced the ... by Testing services - Global Forecast to 2020" ... genetics market is estimated to be worth around $2.5 ... 8-9% during the forecast period of 2014 to 2020 ... animal protein consumption, increasing adoption of advanced genetic technologies ...
(Date:5/28/2015)... 2015 Natera, Inc., a leader in ... in Ultrasound in Obstetrics & Gynecology ... prenatal test (NIPT) can identify a complete molar ... A study based upon data collected in the ... about 1 in 1,000 pregnancies, although other studies ...
Breaking Biology Technology:Global Cytogenetics Market Report 2015-2024 - Technologies, Markets and Companies 2Global Cytogenetics Market Report 2015-2024 - Technologies, Markets and Companies 3Global Alzheimer Disease Report 2015-2024 - New Drugs, Markets and Companies 2Global Alzheimer Disease Report 2015-2024 - New Drugs, Markets and Companies 3Global Animal Genetics (Canine, Equine, Poultry, Porcine, Bovine, and Genetic Material) Market 2015 - Global Forecast to 2020 2First Confirmed Detection of a Complete Molar Pregnancy with Cell-Free DNA Test 2First Confirmed Detection of a Complete Molar Pregnancy with Cell-Free DNA Test 3
... HOUSTON, Feb. 5 In a paper published in the ... Norman L. Sussman, Brendan M. McGuire and James H. Kelly ... device. "There are currently three times as many patients on ... available and few ways to stabilize them if they enter ...
... Feb. 4 /PRNewswire-Asia-FirstCall/ -- BMP Sunstone,Corporation (Nasdaq: BJGP ... will attend the 2009 UBS Global Healthcare Services,Conference, which will ... to 11th. , Management is currently ... 2009, and hold one-on-one meetings the same day. , ...
... U.S. with New Facility and Personnel Additions to Better ... - As part of an ongoing initiative to expand ... has significantly bolstered its data management resources with the ... larger office space, which will be directed by new ...
Cached Biology Technology:Stem Cell Innovations' PluriCells(TM) to Form the Basis of a Liver Assist Device to Support Patients Waiting for Transplant. 2Stem Cell Innovations' PluriCells(TM) to Form the Basis of a Liver Assist Device to Support Patients Waiting for Transplant. 3BMP Sunstone to Present at UBS Global Healthcare Services Conference 2New General Manager, Transition to New Raleigh Offices Highlight Datatrial's Expansion of North American Operations 2
(Date:5/6/2015)... 6, 2015 LifeBEAM, a developer of bio-sensing ... announced today that they will expand their partnership in ... bio-sensing cycling helmet and the first joint project between ... colors in order to give cyclists more style choices ... LifeBEAM and Lazer announced their plan to release a ...
(Date:4/27/2015)... , Apr. 27, 2015 Profile Solutions, Inc. ... and security systems is pleased to announce that Dr ... a consultant and member of its scientific advisory board. ... known as a thought leader in technology-enhanced learning models. ... Group, and has published studies and books focused on ...
(Date:4/17/2015)... Increasing adoption in ... to drive biometric systems market in ... According to a recently released TechSci Research ... biometric systems market in Saudi Arabia ... CAGR of over 22% through 2020. The market ...
Breaking Biology News(10 mins):LifeBEAM and Lazer Announce Expanded Partnership in Bio-Sensing Sports Gear 2LifeBEAM and Lazer Announce Expanded Partnership in Bio-Sensing Sports Gear 3Profile Solutions Names Medicinal Cannabis, Agriculture and Technology Expert Dr. Gerry Bedore to its Scientific Advisory Board 2Profile Solutions Names Medicinal Cannabis, Agriculture and Technology Expert Dr. Gerry Bedore to its Scientific Advisory Board 3Profile Solutions Names Medicinal Cannabis, Agriculture and Technology Expert Dr. Gerry Bedore to its Scientific Advisory Board 4Saudi Arabia Biometric Systems Market to Grow at 22% Through 2020, Says TechSci Research 2Saudi Arabia Biometric Systems Market to Grow at 22% Through 2020, Says TechSci Research 3
... The European eel is on the way to disappearing for ... scientific arguments for suspending all fishing. Despite this situation, Swedish ... management plan by the Swedish Institute for the Marine Environment ... the target that has been set for silver eels capable ...
... the German government to phase out nuclear power by 2022 ... than Germany or Japan, which is still coming to terms ... issue, published by the SETAC journal Environmental Toxicology and ... effects of radiation, using research from the Chernobyl disaster. ...
... a critical part of a human cell,s control system that is ... sensor known as the "gating ring" can open a channel that ... membrane similar to the way a subway turnstile allows people ... in the regulation of crucial bodily activities like blood pressure, insulin ...
Cached Biology News:Will the eel survive its management? 2UCLA scientists identify how major biological sensor in the body works 2UCLA scientists identify how major biological sensor in the body works 3
Yeast tRNA 50 l...
...
... Low volume pipetting is increasingly ... volumes in DNA sequencing and other ... with performance and precision unparalleled in ... documented confirmation of all aspirated,and dispensed ...
... allow for the rapid isolation of DNA from ... slice in the spin filter, centrifuge for 3 ... recovered. There is no need to melt agarose ... can also be used for buffer exchange, primer ...
Biology Products: