The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
T Brown - One of the best experts on this subject based on the ideXlab platform.
-
UNIT 2.9A Southern Blotting
2001Co-Authors: T BrownAbstract:Southern Blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit presents several protocols for DNA transfer to nylon and nitrocellulose membranes, and for calibrating a UV transilluminator to cross-link the transferred DNA to the membrane.
-
Southern Blotting.
Current protocols in molecular biology, 2001Co-Authors: T BrownAbstract:Southern Blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit presents several protocols for DNA transfer to nylon and nitrocellulose membranes, and for calibrating a UV transilluminator to cross-link the transferred DNA to the membrane.
-
APPENDIX 4G Southern Blotting
Current protocols in protein science, 1998Co-Authors: T BrownAbstract:Southern Blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern Blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the Blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid than the basic protocol and can result in more complete transfer. Although the ease and reliability of capillary transfer methods makes this far and away the most popular system for Southern Blotting with agarose gels, it unfortunately does not work with polyacrylamide gels, whose smaller pore size impedes the transverse movement of the DNA molecules. The third alternate protocol describes an electroBlotting procedure that is currently the most reliable method for transfer of DNA from a polyacrylamide gel. Dot and slot Blotting are also described.
-
UNIT 10.6A Southern Blotting
Current protocols in immunology, 1993Co-Authors: T BrownAbstract:Southern Blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern Blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the Blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.
Miranda G S Yap - One of the best experts on this subject based on the ideXlab platform.
-
vector fragmentation characterizing vector integrity in transfected clones by Southern Blotting
Biotechnology Progress, 2010Co-Authors: Wenyu Lin, Rohit Sachdeva, Daniel I C Wang, Miranda G S YapAbstract:The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single-cell clones by Southern Blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single-cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells.
Letizia Foroni - One of the best experts on this subject based on the ideXlab platform.
-
abnormalities of the etv6 gene occur in the majority of patients with aberrations of the short arm of chromosome 12 a combined pcr and Southern Blotting analysis
Leukemia, 1998Co-Authors: H E Oconnor, L M Seckerwalker, Tina Butler, R Clark, S Swanton, Christine J Harrison, Letizia ForoniAbstract:Abnormalities of the ETV6 gene occur in the majority of patients with aberrations of the short arm of chromosome 12: a combined PCR and Southern Blotting analysis
Lawrence F Povirk - One of the best experts on this subject based on the ideXlab platform.
-
ionizing radiation induced dna strand breakage and rejoining in specific genomic regions as determined by an alkaline unwinding Southern Blotting method
International Journal of Radiation Biology, 1995Co-Authors: Roderick T Bunch, D A Gewirtz, Lawrence F PovirkAbstract:A recently developed, combined alkaline unwinding/Southern Blotting assay was utilized to examine DNA damage and repair induced by ionizing radiation within specific large-scale genomic regions. Following treatment of MCF-7 breast tumour cells with 2-10-Gy γ-rays, strand breakage and rejoining were measured in bulk DNA, in the centromeric α-satellite region of chromosome 17, and in the chromatin regions containing the unexpressed β-globin gene and the expressed c-myc oncogene, which is known to be important for growth in the MCF-7 cell line. Damage in both the c-myc and β-globin regions was markedly greater than in either α-satellite or bulk DNA. However, the kinetics of strand break repair were approximately the same in c-myc as in α-satellite or bulk DNA. Surprisingly, the radiomimetic antibiotic bleomycin, which also induces free-radical-mediated strand breakage, showed considerably less heterogeneity of DNA damage among the genomic regions examined than did radiation. The results suggest that actively...
-
a combined alkaline unwinding Southern Blotting assay for measuring low levels of cellular dna breakage within specific genomic regions
Oncology Research, 1992Co-Authors: Roderick T Bunch, D A Gewirtz, Lawrence F PovirkAbstract:A method is described which combines alkaline induced DNA unwinding and Southern Blotting to measure DNA damage occurring in specific genomic regions. Damage induced by gamma-rays at levels as low as 2 Gy was measured in bulk DNA and in a one megabase region surrounding the actively transcribed oncogene, c-myc, as well as in the inactive alpha-satellite DNA of chromosome 17. Although the unwinding kinetics for bulk DNA were consistent with random strand breakage throughout the genome as a whole, measurements at specific loci indicated that the region encompassing c-myc was at least 2-fold more susceptible to damage than either the bulk of the genome or the alpha-satellite region. The results of this study indicate that the combined alkaline unwinding/Southern Blotting assay is a sensitive method for the detection of DNA damage within specific chromatin regions, at biologically relevant doses.
Lefkotheavasiliki Andreou - One of the best experts on this subject based on the ideXlab platform.
-
analysis of dna by Southern Blotting
Methods in Enzymology, 2013Co-Authors: Gary Glenn, Lefkotheavasiliki AndreouAbstract:The purpose of this protocol is to detect specific DNA sequences in a complex sample by hybridization to a labeled probe.
