Optimizing alkaline lysis for DNA plasmid recovery

2003 ◽  
Vol 37 (3) ◽  
pp. 235 ◽  
Author(s):  
Michael Clemson ◽  
William J. Kelly
Keyword(s):  
Alkaline Lysis ◽  
Dna Plasmid ◽  
Plasmid Recovery

scholarly journals Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae.

1990 ◽  
Vol 265 (28) ◽  
pp. 17274-17280
Author(s):  
M Tokunaga ◽  
A Kawamura ◽  
K Kitada ◽  
F Hishinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Killer Toxin ◽  
Linear Dna ◽  
Dna Plasmid

scholarly journals Optimization of a New Non-viral Vector for Transfection: Eudragit Nanoparticles for the Delivery of a DNA Plasmid

2009 ◽  
Vol 8 (6) ◽  
pp. 433-443 ◽  
Author(s):  
M. Gargouri ◽  
A. Sapin ◽  
S. Bouali ◽  
P. Becuwe ◽  
JL Merlin ◽  
...  
Keyword(s):  
Viral Vector ◽  
Dna Plasmid

scholarly journals Keratinocyte growth factor enhances DNA plasmid tumor vaccine responses after murine allogeneic bone marrow transplantation

Blood ◽  
2009 ◽  
Vol 113 (7) ◽  
pp. 1574-1580 ◽  
Author(s):  
Robert R. Jenq ◽  
Christopher G. King ◽  
Christine Volk ◽  
David Suh ◽  
Odette M. Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Keratinocyte Growth Factor ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Effector Cells ◽  
Cd8 Cells ◽  
T Cell Reconstitution ◽  
Dna Plasmid

Abstract Keratinocyte growth factor (KGF), which is given exogenously to allogeneic bone marrow transplantation (allo-BMT) recipients, supports thymic epithelial cells and increases thymic output of naive T cells. Here, we demonstrate that this improved T-cell reconstitution leads to enhanced responses to DNA plasmid tumor vaccination. Tumor-bearing mice treated with KGF and DNA vaccination have improved long-term survival and decreased tumor burden after allo-BMT. When assayed before vaccination, KGF-treated allo-BMT recipients have increased numbers of peripheral T cells, including CD8+ T cells with vaccine-recognition potential. In response to vaccination, KGF-treated allo-BMT recipients, compared with control subjects, generate increased numbers of tumor-specific CD8+ cells, as well as increased numbers of CD8+ cells producing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). We also found unanticipated benefits to antitumor immunity with the administration of KGF. KGF-treated allo-BMT recipients have an improved ratio of T effector cells to regulatory T cells, a larger fraction of effector cells that display a central memory phenotype, and effector cells that are derived from a broader T-cell–receptor repertoire. In conclusion, our data suggest that KGF can function as a potent vaccine adjuvant after allo-BMT through its effects on posttransplantation T-cell reconstitution.

scholarly journals Assessing Quality and Functionality of DNA from Fresh and Archival Dried Blood Spots and Recommendations for Quality Control Guidelines

2007 ◽  
Vol 53 (8) ◽  
pp. 1401-1407 ◽  
Author(s):  
Malin Ida Linnea Sjöholm ◽  
Joakim Dillner ◽  
Joyce Carlson
Keyword(s):  
Dna Extraction ◽  
Multiple Displacement Amplification ◽  
Dried Blood Spots ◽  
Extraction Methods ◽  
Population Based ◽  
Alkaline Lysis ◽  
Screening Programs ◽  
Blood Spots ◽  
Dna Yield ◽  
Chelex 100

Abstract Background: Dried blood spots (DBS) are a convenient and inexpensive method for biobanking. Although many countries have established population-based DBS biobanks from neonatal screening programs, the quality and usefulness of DNA from DBS have not been extensively assessed. Methods: We compared 4 common DNA extraction methods (Qiagen, EZNA, Chelex 100, and alkaline lysis) in a pilot study using fresh DBS with known lymphocyte count. We assessed suitability for multiple displacement amplification (MDA) and subsequent single-nucleotide polymorphism (SNP) analyses. We selected the EZNA method for DNA extraction from archival samples up to 27 years old, stored at room temperature or −20 °C, and SNP analyses were performed after MDA. Results: Extraction using alkaline lysis failed in most tests, and Chelex 100 was unsuccessful in real-time PCR, whereas the EZNA and Qiagen methods were successful by all evaluated quality indices. DNA extraction by EZNA, MDA, and SNP analyses were successful for the archival samples stored at −20 °C. Conclusion: Routine protocols for evaluation of the quality and functional integrity of DNA based on DNA yield, DNA size, and quantification of amplifiable DNA allow use of sufficient template for MDA and successful SNP analyses from both primary DBS extract and MDA product. A single 3-mm disc can yield sufficient DNA for several thousand SNP analyses. DNA from DBS is thus suitable for genetic epidemiology studies.

scholarly journals Protective efficacy of a prime-boost protocol using H5-DNA plasmid as prime and inactivated H5N2 vaccine as the booster against the Egyptian avian influenza challenge virus

2016 ◽  
Vol 60 (03) ◽  
pp. 307-315
Author(s):  
H. A. HUSSEIN ◽  
B. M. AHMED ◽  
S. M. ALY ◽  
A. H. EL-DEEB ◽  
A. A. EL-SANOUSI ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Protective Efficacy ◽  
Challenge Virus ◽  
Dna Plasmid

scholarly journals Horizontal Transfer of Antimicrobial Resistance by Extended-Spectrum β Lactamase-Producing Enterobacteriaceae

2011 ◽  
Vol 3 (01) ◽  
pp. 037-042 ◽  
Author(s):  
Varsha K Vaidya
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Environmental Conditions ◽  
Horizontal Transfer ◽  
Hospital Setting ◽  
Alkaline Lysis ◽  
Antibiotic Resistant ◽  
Extended Spectrum ◽  
Synergy Test

ABSTRACT Background: The purpose of this work was to study the acquisition of new antibiotic-resistant genes carried by extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae via horizontal transfer to understand their rampant spread in the hospitals and in the community. Materials and Methods: A retrospective analysis of 120 ESBL screen-positive isolates of Escherichia coli and Klebsiella pneumoniae, which were subjected to antimicrobial susceptibility testing, was carried out. The Double Disc Synergy Test (DDST) and Inhibitor-Potentiation Disc Diffusion Test (IPDD) were employed for confirmation of ESBL activity. The transferability of the associated antibiotic resistance for amoxicillin, amikacin, gentamicin, cefotaxime and ceftriaxone was elucidated by intra- and intergenus conjugation in Escherichia coli under laboratory as well as under simulated environmental conditions. Transformation experiments using plasmids isolated by alkaline lysis method were performed to study the transferability of resistance genes in Klebsiella pneumoniae isolates. Results : ESBL production was indicated in 20% each of the Escherichia coli and Klebsiella pneumoniae isolates. All the ESBL isolates showed co- resistance to various other groups of antibiotics, including 3GC antibiotics, though all the isolates were sensitive to both the carbapenems tested. Conjugation-mediated transfer of resistance under laboratory as well as environmental conditions at a frequency of 3-4 x 10-5 , and transformation-mediated dissemination of cefotaxime and gentamicin resistance shed light on the propensity of ESBL producers for horizontal transfer. Conclusions: The transfer of resistant markers indicated availability of a large pool of resistance genes in the hospital setting as well as in the environment, facilitating long-term persistence of organisms.

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