An improved method to obtain high molecular weight DNA from purified micro- and macronuclei of Tetrahymena thermophila.
An improved method to obtain high molecular weight DNA from purified macro- and micronuclei of Tetrahymena thermophila is described. Micro- and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (>100 kb) DNA fragments. Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending. We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM). To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods. High molecular weight micro- and macronuclear DNA was obtained using the new protocol.
Chau
M F
MF
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara 93106, USA.
Orias
E
E
D000818:Animals; D016054:DNA, Protozoan / Q000302:isolation & purification* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D008970:Molecular Weight; D016808:Tetrahymena thermophila / Q000235:genetics*
D016054:DNA, Protozoan; D004492:Edetic Acid; D003851:Deoxyribonucleases