Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Examination of BAC DNA Quality and Quantity by Pulsed-Field Gel Electrophoresis

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Pulsed-field gel electrophoresis (PFGE) is the preferred method to measure the size of an insert in a high-capacity vector. After digestion with a restriction enzyme (e.g., PI-SceI or NotI), the DNA fragments are separated by pulsed-field electrophoresis through a 1 agarose gel. Transverse alternating-field electrophoresis (TAFE) resolves DNA fragments between 50 kb and 500 kb in a program in which switch times are ramped from 7 to 50 sec/pulse at 9 V/cm for 2024 h at 15C (Shizuya et al. 1992). Alternatively, a CHEF hexagonal array system can be used with switch times ramping from 1 to 150 sec/pulse at 9 V/cm for 20 h at 15C (Zimmer and Verrinder Gibbins 1997).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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