Naegleria gruberi

Strains, materials and methods

Strain description

Species: Naegleria gruberi, C. Fulton strain NEG-M.
Source: ATCC # 30224

Cell growth and media

Cultured on solid freshwater ameoba medium, including malt and yeast extract (ATCC medium # 997), at 25ºC. Performed by Tom Nerad at ATTC.

Purification of mtDNA

From 0.7 g wet wheight cells, total cellular DNA was extracted. An A+T-rich DNA fraction (mtDNA) was separated from nuclear DNA (nDNA) by equilibrium centrifugation in CsCl-bisbenzimide gradients.

Cloning

Random fragments, generated by nebulization and size fractionation (0.5-1.0 kbp; 1.0-3.0 kbp) were cloned into pFBS, a derivative of Bluescript KS+.
Performed by D. St-Louis (Unit).

Sequencing

Manual DNA sequencing was performed by the dideoxy chain termination method (Sanger et al. 1977), using single-stranded DNA as templates and ³⁵S-dATP as a label. Dried acrylamide gels were autoradiographed and sequences were entered manually. One third of the readings were obtained with a LiCor automatic sequencer, including cycle sequencing (Thermo Sequenase/7-deaza-dGTP, Amersham RPN 2438), using single-stranded and plasmid DNA and a dye-labeled sequencing primer (IRD41).
Performed by Y. Zhu, I. Roewer, and D. St-Louis (Unit).