Capillary gel electrophoresis as a method to determine ligation efficiency.
A capillary gel electrophoresis (CGE) method is described for detection of the formation of circular DNA ligation products as an aid in the prediction of ligated DNA competent cell transformation efficiency. The separation is based upon the differences in the relative migrations of linear and circular DNA molecules of the same size. In CGE, circular ligation products are shifted significantly from linear DNA fragments of comparable size (to 40-42 min from 32-33 min migration time) in the presence of an intercalating dye. CGE separation and detection of circularized DNA can be correlated with transformation efficiencies of > 10(6) colony-forming units (CFU, colonies/micrograms/ml) or the high efficiency desired for phagemid display and cell expression libraries. CGE has several advantages over slab gel electrophoresis: (i) only a minute quantity (approximately 250 CFU or 0.02%) of the total library is sacrificed for analysis, (ii) verification of the circularized ligation products is easier by CGE, and (iii) CGE analysis of ligation success can be accomplished in less than 2 h, prior to transforming competent cells.
Courtney
B C
BC
Applied Pharmacology Branch, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425, USA.
Williams
K C
KC
Bing
Q A
QA
Schlager
J J
JJ
D001435:Bacteriophages / Q000235:genetics; D004247:DNA / Q000032:analysis; D004270:DNA, Circular / Q000032:analysis*; D004278:DNA, Superhelical / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005822:Genetic Vectors; D007364:Intercalating Agents; D008025:Ligases; D014170:Transformation, Genetic
D004270:DNA, Circular; D004278:DNA, Superhelical; D007364:Intercalating Agents; D004247:DNA; D008025:Ligases