Monodisperse DNA restriction fragments I. Synthesis and characterization.
We present a convenient and low-cost method to prepare milligram amounts of completely monodisperse DNA restriction fragments in a physico-chemical laboratory setting to study (in part II) the effect of limited flexibility on the concentration dependent sedimentation velocity. Four fragments of 200, 400, 800, and 1600 bp were designed to span a range of 1-11 persistence lengths. The fragments were synthesized by cloning fragments of controlled lengths obtained by PCR into bacterial plasmid DNA. The constructs were amplified in large-scale bacterial cultures from which the fragments were obtained by a modified alkaline lysis procedure and subsequent digestion with EcoRV. A method is presented to isolate the DNA from the digestion mixture using horizontal agarose-slab gels and agarose columns in a home-built preparative gel electrophoresis set-up. We show that a combination of optical absorbance readings, ethidium bromide fluorescence, and hyperchromicity measurements allows assessment of both the purity of the DNA solutions and the fraction of double-stranded DNA.
Planken
Karel L
KL
Van't Hoff Laboratory for Physical and Colloid Chemistry, Debye Institute, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. k.l.planken@chem.uu.nl
Koenderink
Gijsberta H
GH
Roozendaal
Ramon
R
Philipse
Albert P
AP
D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004996:Ethidium; D005456:Fluorescent Dyes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
D005456:Fluorescent Dyes; D012996:Solutions; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D004996:Ethidium