[The study of optimal conditions of electroporation in Escherichia coli DH10B strain].
In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.
Zhang
Yang
Y
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Wang
Zhi-Qiang
ZQ
Liu
Bin
B
Zhang
Xiao-Jun
XJ
Jiang
Feng
F
Xiang
Jian-Hai
JH
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics*; D014170:Transformation, Genetic
D004269:DNA, Bacterial