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KMID : 0357319920270060525
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Journal of the Korean Society for Microbiology
1992 Volume.27 No. 6 p.525 ~ p.536
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Studies on the Transformation Efficiency Factors of E. coli by pBR 322 DNA
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Abstract
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To screen for very rare dDNA clones a cDNA library consisting of at elast 5×10E6 independetn clones ofter need to be constructed with a limite amound of mRNA. This is why a highly efficient transformation methods needed in construction of cDNA
libraries with a miniumum expenditure of mRNA.
PBR 322 DNA was isolated and purified and then used to transform Escherichia coli with rec A genotype. Optimal conditions for the transformation of E. coli prepared by calcium chloride(CaCl2) solution, resistance by trasnsformed cells were
assayed
by
selective medium and transfered plasmid DNA was identified by agarose gel electrophoresis.
@ES Obtained results was summerized as follows:
@EN 1. In CaCl2 treatment, maximal transformation was achieved when cells were exposed to 0.1M CaCl2 solution at 0℃ for 24 hours were 4 times more efficient in transformation then those without low temperature treatment.
2. Among transformation bufers tested, 10mM N-(2-hydroxymethyl)-piperazine-n'-1-ethane-sulfonic acid(Hepes) yielded a transformation efficiency of 1×10E8 transformant/㎍ plasmid NDA. It is 10 times more efficient than potassium acetate and 2.5
times
more transformable than 30(N-morpholino) propanesulfonic acid(MOPS).
3. Incubation at 25℃ was 9 times more transformable than incubation at 37℃when treated with transformation buffer for SEM(TB) solution. Use of transformation buffer for frozen storage of competen cells(FSB) solution, however, resulted in only
low
competence even after incubation at 25℃.
4. Frozen competent cells treated with TB solution after long term storage in the presence of DMSO(70%) were more stable than when treated with FSB solution or CaCl2 solution.
Consequently, SEM protocol for preparing competent E. coli is extremely efficient for plasmid transformation and the cells cal be stored at -70℃ for than a month. These competne cells are suited for construction of cDNA library.
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