Pulsed field gel electrophoresis on single murine hemopoietic colonies.
We report a technique which allows for the direct molecular analysis of single whole murine hemopoietic colonies by pulsed field gel electrophoresis (PFGE). Murine bone marrow cells were plated out in semi-solid agarose and gave rise to macroscopic colonies after 11 days in culture. Single colonies were excised from the agarose using a sterile blade and embedded without further manipulation in molten low-melting-temperature agarose. The leucocyte DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is afforded by this method to achieve a hybridization signal with a single copy probe. This method will make PFGE directly applicable to the clonal analysis of chromosomal aberrations in hemopoietic stem and progenitor cells.
Boultwood
J
J
Department of Haematology, John Radcliffe Hospital, Headington, Oxford, UK.
Thompson
M
M
Fidler
C
C
Lorimore
S A
SA
Lewis
M S
MS
Wainscoat
J S
JS
Wright
E
E
D000818:Animals; D001853:Bone Marrow / Q000737:chemistry; D001854:Bone Marrow Cells; D003114:Colony-Forming Units Assay; D004247:DNA / Q000032:analysis* / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D012685:Sepharose
D004247:DNA; D012685:Sepharose