scholarly journals IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e107907 ◽  
Author(s):  
Jiancai Wang ◽  
Ronghua Xu ◽  
Aizhong Liu
Keyword(s):  
Recombinant Dna ◽  
Dna Preparation ◽  
Cloning Method ◽  
Directional Cloning

Improvement of TA Cloning Method to Facilitate Direct Directional Cloning of PCR Products

2014 ◽  
Vol 565 ◽  
pp. 3-8 ◽  
Author(s):  
Ji Gang Li ◽  
Guo Ying Han ◽  
Xiu Min Li ◽  
Jiao Jiao Sun ◽  
Ke Jing Song ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Pcr Primers ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reaction Products ◽  
Major Disadvantage ◽  
Cloning Method ◽  
Directional Cloning ◽  
Pcr Products ◽  
Green Fluorescent

Directional cloning is a prerequisite for the construction of expression vectors in molecular biology laboratories. Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional. Here we reported a novel PCR products cloning vector with one deoxythymidine overhang and one deoxycytidine overhang at two 3'-ends respectively. With the choice of nucleotides of 5'-ends of PCR primers, PCR products can be cloned to this vector both directly and directionally. The feasibility and efficacy of this cloning method were confirmed by using a pET-17b derivative vector and a green fluorescent protein gene (EGFP) and a red fluorescent protein reporter (Ds-Red) gene. This cloning strategy may be useful in the high-throughput construction of expression vectors and could be viewed as an interesting improvement of existing TA cloning method.

scholarly journals One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies

2012 ◽  
Vol 78 (15) ◽  
pp. 5440-5443 ◽  
Author(s):  
Jae-Yeon Jeong ◽  
Hyung-Soon Yim ◽  
Ji-Young Ryu ◽  
Hyun Sook Lee ◽  
Jung-Hyun Lee ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Dna Polymerase ◽  
Room Temperature ◽  
Cost Effective ◽  
Time Saving ◽  
Ligation Independent Cloning ◽  
Effective Time ◽  
Cloning Method ◽  
Directional Cloning ◽  
One Step

ABSTRACTWe developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

A Multicenter Study of Recombinant Factor VIII (RecombinateTM) in Previously Treated Patients with Hemophilia A

1997 ◽  
Vol 77 (04) ◽  
pp. 660-667 ◽  
Author(s):  
G C White ◽  
S Courter ◽  
G L Bray ◽  
M Lee ◽  
E D Gomperts ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Recombinant Dna ◽  
Home Treatment ◽  
Pharmacokinetic Parameters ◽  
Pharmacokinetic Studies ◽  
Open Label ◽  
Treat Ment ◽  
Previously Treated Patients ◽  
Bleeding Episodes

SummaryA prospective, open-label multicenter investigation has been conducted to compare pharmacokinetic parameters of recombinant DNA-derived FVIII (rFVIII) and plasma-derived FVIII concentrate (pdFVIII) and to assess safety and efficacy of long-term home-treat- ment with rFVIII for subjects with hemophilia A. Following comparative in vivo pharmacokinetic studies, 69 patients with severe (n = 67) or moderate (n = 2) hemophilia A commenced a program of home treatment using rFVIII exclusively for prophylaxis and treatment of all bleeding episodes for a period of 1.0 to 5.7 years (median 3.7 years). The mean in vivo half-lives of rFVIII and pdFVIII were both 14.7 h. In vivo incremental recoveries at baseline were 2.40%/IU/kg and 2.47%/IU/kg, respectively (p = 0.59). The response to home treatment with rFVIII was categorized as good or excellent in 3,195 (91.2%) of 3,481 evaluated bleeding episodes. Thirteen patients received rFVIII for prophylaxis for twenty-four surgical procedures. In all cases, hemostasis was excellent. Adverse reactions were observed in only 13 of 13,591 (0.096%) infusions of rFVIII; none was serious. No patient developed an inhibitor to r FVIII.

Enzymatic Synthesis of 15N-L-aspartic Acid Using Recombinant Aspartase from Escherichia Coli K12

2008 ◽  
Vol 59 (11) ◽  
Author(s):  
Iulia Lupan ◽  
Sergiu Chira ◽  
Maria Chiriac ◽  
Nicolae Palibroda ◽  
Octavian Popescu
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Enzymatic Synthesis ◽  
Large Scale ◽  
Recombinant Dna ◽  
Industrial Enzymes ◽  
Recombinant Dna Technology ◽  
Bacterial Fermentation ◽  
Natural Protein ◽  
Novel Method

Amino acids are obtained by bacterial fermentation, extraction from natural protein or enzymatic synthesis from specific substrates. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to enzymatic synthesis of amino acids. Aspartase (L-aspartate ammonia-lyase) catalyzes the reversible deamination of L-aspartic acid to yield fumaric acid and ammonia. It is one of the most important industrial enzymes used to produce L-aspartic acid on a large scale. Here we described a novel method for [15N] L-aspartic synthesis from fumarate and ammonia (15NH4Cl) using a recombinant aspartase.

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