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:9/1/2015)... MILWAUKEE , Sept. 1, 2015 ... Eric Floyd , Ph.D., MBA as Chief ... (DLSS).  In this newly created role, Dr. Floyd ... well as a portfolio of pre-commercial and post ... medical affairs and medical communications.  Dr. Floyd brings ...
(Date:9/1/2015)... ... September 01, 2015 , ... A new host-based therapy for ... the Keck Graduate Institute and its collaborators. The discovery has the potential to ... online on August 27 by Scientific Reports, an open access research journal from ...
(Date:8/31/2015)... 31, 2015 /PRNewswire/ - BIOREM Inc. (TSXV: BRM) ("Biorem" or ... orders are for air emission abatement projects in ... a large order for a municipality in the ... a competitor,s system that failed to meet the customer,s requirements. This ... said Derek S. Webb , President and Chief Executive ...
(Date:8/31/2015)... 31, 2015 Research and Markets ( http://www.researchandmarkets.com/research/53wxp3/israel_biomedical ... Biomedical Sensors Market - Growth, Trends & Forecasts (2014-2020)" ... Israel Biomedical Sensors market is estimated at $0.19 billion ... period 2014-2020 The near future will bring ... of each individual. These sensors can be set to ...
Breaking Biology Technology:Dohmen Life Science Services Appoints Eric Floyd, Ph.D. as Chief Science Officer 2Dohmen Life Science Services Appoints Eric Floyd, Ph.D. as Chief Science Officer 3Scientists Discover New Use for an Old Therapy Inhibiting Deadly Pathogens Including Ebola and Anthrax 2Scientists Discover New Use for an Old Therapy Inhibiting Deadly Pathogens Including Ebola and Anthrax 3Scientists Discover New Use for an Old Therapy Inhibiting Deadly Pathogens Including Ebola and Anthrax 4Israel Biomedical Sensors Market 2015 - Growth, Trends & Forecasts to 2020 2
... Presentation Scheduled for Monday, March 17th at 11:30am ET, ... KERX ) today announced that I. Craig Henderson, ... Vice President,Medical Affairs, are scheduled to present at the ... The presentation, which will take place on Monday, ...
... Phase 2 Clinical Trial in ... ... today financial results for the year ended December,31, 2007, and highlighted the ... ), During 2007, the Company achieved the following corporate milestones:, ...
... for ... cars, phones, computers, ARGONNE, Ill., ... Laboratory and Toda Kogyo Corp. (Toda) of Japan have,reached a ... patented composite cathode materials for lithium-ion,batteries, which result in longer-lasting, ...
Cached Biology Technology:Keryx Biopharmaceuticals to Present at the Cowen and Company 28th Annual Health Care Conference 2Nventa Biopharmaceuticals Reports Full Year 2007 and Fourth Quarter Financial Results 2Nventa Biopharmaceuticals Reports Full Year 2007 and Fourth Quarter Financial Results 3Nventa Biopharmaceuticals Reports Full Year 2007 and Fourth Quarter Financial Results 4Nventa Biopharmaceuticals Reports Full Year 2007 and Fourth Quarter Financial Results 5Nventa Biopharmaceuticals Reports Full Year 2007 and Fourth Quarter Financial Results 6Nventa Biopharmaceuticals Reports Full Year 2007 and Fourth Quarter Financial Results 7Argonne's Lithium-Ion Battery Technology to be Commercialized by Japan's Toda Kogyo 2Argonne's Lithium-Ion Battery Technology to be Commercialized by Japan's Toda Kogyo 3
(Date:8/20/2015)... , Aug. 20, 2015   Sensory, ... on improving the user experience and security of ... technologies, today announced that its TrulySecure™ ... authentication software to be FIDO Certified™. ... for compliance with the FIDO UAF (Universal Authentication ...
(Date:8/19/2015)... HAUPPAUGE, N.Y. , Aug. 19, 2015 /PRNewswire/ ... announced that it has entered into a Definitive ... intellectual property, of iris authentication market leader EyeLock ... transaction, VOXX will have a controlling interest in ... is subject to completion of due diligence.  Expanding ...
(Date:8/18/2015)... Research and Markets ( http://www.researchandmarkets.com/research/9dmm6l/global_biometric ) ... Biometric Authentication & Identification Market: Focus On Modality, ... report to their offering. ... is expected to grow at an estimated CAGR ... over $25 billion (approximately) by 2020 as per ...
Breaking Biology News(10 mins):TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 2TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 3TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 4TrulySecure From Sensory Becomes First Face and Voice Biometrics Technology to be FIDO UAF Certified 5VOXX International Corporation Signs Definitive Agreement To Acquire The Assets And A Controlling Interest Of Biometric Leader EyeLock 2VOXX International Corporation Signs Definitive Agreement To Acquire The Assets And A Controlling Interest Of Biometric Leader EyeLock 3VOXX International Corporation Signs Definitive Agreement To Acquire The Assets And A Controlling Interest Of Biometric Leader EyeLock 4VOXX International Corporation Signs Definitive Agreement To Acquire The Assets And A Controlling Interest Of Biometric Leader EyeLock 5Global Biometric Authentication & Identification Market 2015: Focus On Modality, Motility & Application - Estimation & Forecasts to 2020 for the $25 Billion Industry 2
... The Laboratory for Virology and Experimental Chemotherapy (Rega ... at K.U.Leuven will receive a total of 2.8 million euro ... to the search for possible new medication for the treatment ... ,breakbone fever, is widespread in tropical and subtropical regions. As ...
... international team of researchers led by geneticist Jonathan Sebat, Ph.D., ... on human chromosome 16 that substantially increases risk for schizophrenia. ... a copy number variant (CNV). CNVs are areas of the ... individuals. The CNV is located in a region referred to ...
... Calif., Oct. 23 GenVault Corporation, the leader in ... identification, today announced that David Wellis, Ph.D., GenVault,s President ... Conference taking place on October 26th and 27th in ... will provide an overview of the company together with ...
Cached Biology News:Wellcome Trust funds dengue fever research in Leuven 2CSHL-led team discovers rare mutation dramatically increasing schizophrenia risk 2GenVault Corporation to Present at the 2009 BIOCOM Investor Conference 2
...
Anti-human L-Ficolin, Clone GN4, Monoclonal Antibody...
Jak3 Antibody Ship: Hot Store: -20 C...
B3GALT2 Immunogen: B3GALT2 (NP_003774, 324 a.a. ~ 423 a.a) partial recombinant protein with GST tag. Storage: -20 C, Avoid Freeze/Thaw Cycles *Available for distribution in the US only...
Biology Products: