Chen H, Nelson R S, Sherwood J L
Oklahoma State University, Stillwater.
Biotechniques. 1994 Apr;16(4):664-8, 670.
Freeze-thaw transformation provides a simple and rapid method to transform Agrobacterium tumefaciens directly with plasmid DNA. Competent A. tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transformed with four to nine plasmids differing in size, size of insert and in some cases sensitivity to antibiotics. A threefold to fourfold increase in transformed colonies per microgram of DNA was obtained by freezing cells with liquid nitrogen vs. dry ice/ethanol. Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low concentration of appropriate antibiotics prior to plating resulted in a ninefold increase in colonies obtained compared with the procedure of freezing cells in dry ice/ethanol without the incubation period in the low concentration of antibiotics prior to plating. Restriction fragments of the expected sizes from the plasmids indicated that the procedural modifications did not cause apparent recombinations in the region of the inserts.
冻融转化法提供了一种简单快速的方法，可直接用质粒DNA转化根癌农杆菌。用四到九种在大小、插入片段大小以及某些情况下对抗生素敏感性不同的质粒，对LBA4404、GV3850和EHA101菌株的感受态根癌农杆菌细胞进行转化。与用干冰/乙醇冷冻细胞相比，用液氮冷冻细胞可使每微克DNA转化菌落数增加三到四倍。先将细胞用液氮冷冻，然后在低浓度的合适抗生素中孵育转化细胞，再进行平板接种，与在干冰/乙醇中冷冻细胞且在平板接种前不经过低浓度抗生素孵育期的方法相比，获得的菌落数增加了九倍。来自质粒的预期大小的限制性片段表明，程序上的修改在插入片段区域未引起明显的重组。
