TY - JOUR
T1 - Gel electrophoretic analysis of bacteriophage assembly intermediates in bacteriophage plaques.
AU - Serwer, P.
AU - Hayes, S. J.
AU - Watson, R. H.
AU - Khan, S. A.
PY - 1995
Y1 - 1995
N2 - To increase the efficiency with which the phenotype of bacteriophage mutants is determined by gel electrophoresis, procedures are developed here for the preparation of the contents of bacteriophage plaques for gel electrophoresis. During the formation of plaques, the plaque-supporting upper layer gel is changed from the traditional agar gel to a gel made of a mixture of low-melt agaroses; the lower layer gel is eliminated. To extract particles from plaques, the plaque-supporting gel is disintegrated by both shaking and raising the temperature to 39-43 degrees C. During shaking, the gel is broken to domains that are 5-30 microns in diameter. After extraction, the contents of plaques are subjected to two electrophoretic analyses: (1) Nondenaturing agarose gel electrophoresis is performed after treatment with DNase. This procedure reveals both mature bacteriophage and immature capsids. (2) Nondenaturing agarose gel electrophoresis is performed after release of DNA from DNase-treated capsids. This latter procedure reveals both completely packaged (mature length) DNA and incompletely packaged (shorter than mature length) DNA. The amount of mature length DNA released per 2-3 mm plaque is 10-60 ng. In agreement with results previously obtained in liquid culture, most incompletely packaged DNA has the right, but not the left, mature T7 DNA end.
AB - To increase the efficiency with which the phenotype of bacteriophage mutants is determined by gel electrophoresis, procedures are developed here for the preparation of the contents of bacteriophage plaques for gel electrophoresis. During the formation of plaques, the plaque-supporting upper layer gel is changed from the traditional agar gel to a gel made of a mixture of low-melt agaroses; the lower layer gel is eliminated. To extract particles from plaques, the plaque-supporting gel is disintegrated by both shaking and raising the temperature to 39-43 degrees C. During shaking, the gel is broken to domains that are 5-30 microns in diameter. After extraction, the contents of plaques are subjected to two electrophoretic analyses: (1) Nondenaturing agarose gel electrophoresis is performed after treatment with DNase. This procedure reveals both mature bacteriophage and immature capsids. (2) Nondenaturing agarose gel electrophoresis is performed after release of DNA from DNase-treated capsids. This latter procedure reveals both completely packaged (mature length) DNA and incompletely packaged (shorter than mature length) DNA. The amount of mature length DNA released per 2-3 mm plaque is 10-60 ng. In agreement with results previously obtained in liquid culture, most incompletely packaged DNA has the right, but not the left, mature T7 DNA end.
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M3 - Article
C2 - 7599257
AN - SCOPUS:0029199032
VL - 4
SP - 211
EP - 217
JO - Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society
JF - Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society
SN - 0954-6642
IS - 4
ER -