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

d by Dr. John McCafferty (Cambridge Antibody Technology).
Two DNA fragments (here designated as fragments A and B) had to be joined at a common unique Alu I site and ligated with a fd-tet DNA backbone at Apa LI and Bam HI restriction sites (Figure 1). Both insert fragments were obtained by cleavage of cloned DNA with the endonucleases Apa LI, Bam HI, and Alu I. The Apa LI/Alu I fragment of insert A and the Alu I/Bam HI fragment of insert B were purified after separation on an agarose gel. One microgram of DNA in a total volume of 10 l was used in the conventional ligation reactions, with a molar ratio of phage DNA to insert A and insert B of 1:5:5. Standard ligations of the two insert fragments together with appropriately cleaved fd-tet DNA were performed for 24 h at 14 C. Our optimized procedure was comprised of two temperature steps: first, the blunt-end ligation of the insert fragments A and B for 18 h at 26C; second, the cohesiveended vector DNA was added, and the ligation continued for an additional 6 h at 14C [3]. The protocol for the Rapid DNA Ligation Kit was modified in accordance with our optimized conventional procedure. The blunt-end ligation mixture (290 ng DNA in a total volume of 20 l) was ligated for 30 min at 26C. Next, 710 ng vector DNA was added in a volume of 1 l with further incubation for 10 min at room temperature.

The ligation mixtures were then cooled to 0C without heat-inactivation of the ligase, and one third of each reaction was used to transform DH5a competent cells, which were plated on 140 mm Petri dishes containing 30 mg tetracycline/ml LB medium. After a 20 h incubation period, colonies were counted, and doublestranded phage DNA of a representative number purified with the High PureTM Plasmid Isolation Kit. The DNA was screened by restriction analysis with the
'"/>

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:10/20/2014)... MENLO PARK, Calif. , Oct. 20, 2014 ... announced today that the Company has signed a ... Institute of Regenerative Medicine (CIRM), effective October 1, ... of Clinical Development payments and the release of ... $14.3 million CIRM grant award for clinical development ...
(Date:10/20/2014)... 20, 2014 PureTech , a science ... healthcare problems, announced today the closing of a ... from Invesco Perpetual, a $120 Billion group of ... PureTech,s existing pipeline forward and to advance new ... scientific creativity to really go for the big ...
(Date:10/19/2014)... 2014 The Latin American hardware encryption ... Latin America with analysis and forecast of revenue. This ... is expected to reach $2,366.8 million by 2018, at ... Browse through the TOC of the Latin American hardware ... in-depth analysis provided. It also provides a glimpse of ...
(Date:10/19/2014)... 2014 The Asian Automatic patient billing ... analysis and forecast of revenue. The Automatic patient billing ... $463.9 million by 2018, at a developing CAGR of ... TOC of the Asian Automatic patient billing market, to ... also provides a glimpse of the segmentation of this ...
Breaking Biology Technology:Asterias Biotherapeutics Announces Notice of Grant Award with CIRM for Phase 1/2a Clinical Trial of AST-OPC1 in Complete Cervical Spinal Cord Injury 2Asterias Biotherapeutics Announces Notice of Grant Award with CIRM for Phase 1/2a Clinical Trial of AST-OPC1 in Complete Cervical Spinal Cord Injury 3Asterias Biotherapeutics Announces Notice of Grant Award with CIRM for Phase 1/2a Clinical Trial of AST-OPC1 in Complete Cervical Spinal Cord Injury 4PureTech Announces Expansion with Oversubscribed $55M Funding Round 2PureTech Announces Expansion with Oversubscribed $55M Funding Round 3PureTech Announces Expansion with Oversubscribed $55M Funding Round 4PureTech Announces Expansion with Oversubscribed $55M Funding Round 5PureTech Announces Expansion with Oversubscribed $55M Funding Round 6The Latin American hardware encryption display market is expected to reach $2,366.8 million by 2018 - New Report by MicroMarket Monitor 2The Latin American hardware encryption display market is expected to reach $2,366.8 million by 2018 - New Report by MicroMarket Monitor 3The Asian Automatic patient billing market is estimated to grow to around $463.9 million by 2018 - New Report by MicroMarket Monitor 2The Asian Automatic patient billing market is estimated to grow to around $463.9 million by 2018 - New Report by MicroMarket Monitor 3
... past few years, many companies have been downsizing their ... name speculation market had cooled. There has not been, ... saw a 20% increase in the number of domain ... , ,The domain name market has been reinvigorated due ...
... Committee voted unanimously to authorize an audit of troubled ... 6-0 vote came after several hours of testimony on ... state has lost millions of dollars that cannot be ... been plagued by varying problems, including delays in implementation, ...
... upon us as I write these words! This year's theme, ... in the Midwest from medical devices to diagnostics to drugs ... ,Although all kinds of records are being broken in terms ... on the final attendance number, but the pre-registration number floating ...
Cached Biology Technology:Renew your domain name! 2Renew your domain name! 3Renew your domain name! 4Audit of multi-million dollar state IT spending will proceed 2Let the olympics of biotechnology in the Midwest begin! 2Let the olympics of biotechnology in the Midwest begin! 3Let the olympics of biotechnology in the Midwest begin! 4
(Date:10/16/2014)... have challenged conventional thinking on how the bowel lining ... for how bowel cancer starts. , The researchers produced ... regenerating the ,crypts, that are a feature of the ... in bowel cancer development, a controversial finding as scientists ... Using 3D imaging technologies, Dr Chin Wee Tan and ...
(Date:10/16/2014)... Northridge, Calif. (October 16, 2014) – Post-menopausal women ... effect on hormone levels or genital bleeding, after ... of an investigational fermented soy germ-based nutritional supplement ... according to a new peer-reviewed pilot study reported ... Society (NAMS) annual scientific meeting. , "These ...
(Date:10/15/2014)... risk from strains of influenza virus increases with the ... become complacent that the most substantial threats have been ... pandemics arise when a new virus strain – against ... spreads in the human population. There have been five ... of which – the 1918 Spanish Flu – cost ...
Breaking Biology News(10 mins):Cryptic clues drive new theory of bowel cancer development 2S-equol supplements associated with improved measures of reproductive health in postmenopausal women 2Scientists 'must not become complacent' when assessing pandemic threat from flu viruses 2
... identified the first common gene variants associated with an ... for heart attack, stroke and kidney failure. The report ... Genetics identifies variants in genes for proteins involved ... influence blood pressure levels, an association previously seen in ...
... disease, red tides and other marine environmental issues ... a source for pharmaceuticals. These chemicals function as ... be usable in human health care. ... The Medical University of South Carolina and North ...
... one-sixth of the world,s population -- nearly one billion people, ... basis? If you happen to be one of 15 ... your sleeves and design a $5, hand-held device to sanitize ... began as a class project, it has since become a ...
Cached Biology News:Common gene variants increase risk of hypertension, may lead to new therapies 2Breaking the barrier: Discovery of anti-resistance factors and novel ocean drugs 2Breaking the barrier: Discovery of anti-resistance factors and novel ocean drugs 3Iowa student engineers develop hand-held water sanitizer for a thirsty world 2
Mol wt: average mol wt12,360.96 Da by calculation...
... Grace's medium were developed in several laboratories ... of these modifications were developed by Dr. ... derived from the cabbage looper,Tricoplusia ni. This ... The medium when properly supplemented has been ...
... Disposable. Virgin, optically clear polystyrene ... and alphanumeric coordinates for cell counting. ... number is a new product number, ... number. If showing no availability yet, ...
... This CLS number is a ... easily match Cornings product number. ... please order under the old ... customer service for assistance. ...
Biology Products: