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Separation of large DNA molecules with high voltage pulsed field gel electrophoresis
Author(s) -
Wagner Lauren,
Lai Eric
Publication year - 1994
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501501161
Subject(s) - electrophoresis , voltage , gel electrophoresis , chromatography , resolution (logic) , dna , analytical chemistry (journal) , electric field , high voltage , materials science , chemistry , physics , biochemistry , computer science , quantum mechanics , artificial intelligence
We have developed two high voltage (3 kV and 10 kV) high speed pulsed field gel electrophoresis systems for the separation of DNA as large as 460 kbp. These systems enable us to combine the rapid speed of high voltages and the separation power of pulsed field electrophoresis to achieve high resolution and short run durations. We found that large DNA fragments can be separated at voltage gradients much higher than commonly used. Yeast chromosomes as large as 460 kbp can be separated in 4 h at 20 V/cm and 1–50 kbp DNA can be rapidly separated in 30 min at 55 V/cm. This is 25 times faster in mobility for the separation of relatively small DNA fragments (< 50 kbp). We have also found an inverse relationship between the voltage applied and the size separation limit at that particular voltage gradient (55 V/cm limits the separation to 50 kbp while 20 V/cm can separate up to 460 kbp). Depending on the size range, DNA can be separated 8‐ to 25‐fold faster and with better resolution than existing electrophoretic systems.