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:7/25/2014)... Muncie, IN (PRWEB) July 25, 2014 ... Administration (FAA) has granted a 60-day extension for the ... of the Special Rule for Model Aircraft established by ... Act of 2012. The 60-day extension establishes the new ... the United States Department of Transportation/FAA notice published in ...
(Date:7/24/2014)... Calabasas, CA (PRWEB) July 24, 2014 ... advancing the Global Supply Chain of the biotech ... annual SCM Innovation Awards, brought to you by ... premiere global organization of supply chain management professionals ... be given to stakeholders of the Biotech industry ...
(Date:7/24/2014)... Security forces worldwide rely on sophisticated equipment, trained ... other public areas against terrorist attacks. A revolutionary ... about to make their job much easier. , ... Patolsky of Tel Aviv University ,s School of ... developed by the Herzliya company Tracense, picks up ...
(Date:7/24/2014)... prefer to settle into equilibriuma state of unchanging ... realm of non-equilibrium conditions where new possibilities lie. ... phases, such as temperature fluctuations, freezing and melting, ... their body temperature, airplanes to fly, and the ... But even though these conditions exist naturally and ...
Breaking Biology Technology:FAA Grants Comment Extension at the Request of AMA 2Call for Submission: BSMA’s Supply Chain Management Innovation Awards 2Call for Submission: BSMA’s Supply Chain Management Innovation Awards 3Nano-sized chip 'sniffs out' explosives far better than trained dogs 2New approach to form non-equilibrium structures 2
(Date:7/24/2014)... YORK, NY (July 24, 2014) Scientists at ... Institute are one step closer to creating a ... a patient,s own cells. , For the ... (iPS) cells lines from skin samples of patients ... developed an accelerated protocol to induce these stem ...
(Date:7/24/2014)... of native animal species for food or commercial food ... factor in the decline of many species of primates ... that more than half of the species being consumed ... "By surveying not only the meat made available for ... the forest by hunters and brought to villages for ...
(Date:7/24/2014)... of 3.5 billion years of evolutionary trial and error, ... it may be reaching a tipping point. , In ... data published in Science , an international team ... animals is contributing to what appears to be the ... event. , Since 1500, more than 320 terrestrial vertebrates ...
Breaking Biology News(10 mins):NYSCF scientists one step closer to cell therapy for multiple sclerosis patients 2Stanford biologist warns of early stages of Earth's 6th mass extinction event 2Stanford biologist warns of early stages of Earth's 6th mass extinction event 3
... human evolution follows a progressive trajectory is one of ... is often expressed in popular media by showing cavemen ... a notable exception). But is this assumption correct? ... In a paper published in the ...
... stop the spread of a deadly fungus that is threatening ... a new mathematical model. , White-nose syndrome, which is estimated ... year period, is probably caused by a newly discovered cold-adapted ... WNS is passed from bat to bat and concludes that ...
... and warms the world,s oceans with carbon emissions, the harder ... That,s the blunt message from a major new study ... and global warming will combine with local impacts like overfishing ... when they are struggling to survive. Modelling by a ...
Cached Biology News:Earliest humans not so different from us, research suggests 2Earliest humans not so different from us, research suggests 3Culling can't control deadly bat disease 2Acid oceans demand greater reef care 2
... System provides tightly regulated control of exogenous ... capable of repeated on/off expression kinetics. This ... highly specific interaction between the GenoStat Ligand ... Gene expression levels are easily manipulated higher ...
... added in Dec 2005 , Quantitative ... Detection from as little as 25 pg ... available for hundreds of mammalian miRNAs , ... , Ideal for validating expression profiling ...
Recommend to use at up to 5000X dilutions for cell culture (e.g. Add 1 ul to 1 ml of culture medium.)...
... Molecular Weight Markers, 37 kBq, 1 uCi. ... determination of the sample on Western blots.The ... band migration in the gel and on ... version without Rainbow-labeling is 0.37-3.7 MBq/mg (10-100 ...
Biology Products: