Optimization of routine transformation of Escherichia coli with plasmid DNA.
Methods to optimize resources and transformation efficiency of routine daily transformations of DH1 Escherichia coli prepared by three calcium chloride methods were investigated and compared with polyethylene glycol and Hanahan methods. The benefit of a heat-shock step, a preplating incubation step to allow expression of antibiotic resistance, use of log phase bacteria and prolonged storage of bacteria were investigated using pBR322 and pUC18 plasmid DNAs. Bacteria prepared by CaCl2 methods consistently gave efficiencies of 4 x 10(6) transformants/microgram of plasmid DNA or better and were overall the most labor- and resource-efficient methods. Use of log phase bacteria, a heat shock and an incubation step were found to be beneficial for freshly prepared bacteria for all methods. Prolonged storage of up to 30 days of bacteria prepared by the CaCl2 methods was beneficial, resulting in a sustained increase in transformation efficiency when selection was by ampicillin but not when by tetracycline resistance. Also found when using bacteria stored three days or longer was an increased transformation efficiency of stationary vs. log phase bacteria and an unchanged or even increased efficiency when the preplating incubation step was omitted. The Hanahan methods were the most labor and resource intensive and routinely gave efficiencies of 2 x 10(7). Higher efficiencies of 10(8) were obtained only with repeated trial and error and were not consistently reproducible. The polyethylene glycol method consistently gave efficiencies of 2 x 10(7), and bacteria could easily be prepared daily or frozen with a minimal decrease in efficiency.
Huff
J P
JP
Section of Rheumatology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612.
Grant
B J
BJ
Penning
C A
CA
Sullivan
K F
KF
D000668:Ampicillin Resistance; D002122:Calcium Chloride; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005821:Genetic Techniques; D006358:Hot Temperature; D010957:Plasmids; D013753:Tetracycline Resistance; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D002122:Calcium Chloride