Agarose gels, relaxation

Fig. 8.1. (a) DNA forms form I, supercoiled form II, relaxed form III, linear, (b) Schematic representation of agarose gel electrophoresis of forms I, II and III. 

Figure 9.152 Profile of separation of relaxed DNA from supercoiled plasmid pBR329 on a DEAE-NPR column. The reaction was carried out with 1 unit of calf thymus topoisomerase I. Twenty microliters of the reaction mixture was applied on the column and eluted with a linear gradient of 0.5 to 0.65 M NaCl for 30 minutes. DNAs from peaks a and b were collected manually and analyzed by electrophoresis on a 1% agarose gel after ethanol precipitation. Inset O.C., open circular S.C., supercoiled. (From Onishi et al., 1993.)...

Fig. 9. Positive supercoiling of SSVl DNA. Panels A and B show two-dimensional agarose gel electrophoresis of SSVl DNA isolated from cells of Sulfolobus shibatae, (A) before and (B) after UV induction (for methods see refs. [39,98]). The left-hand branch of the arch visible in A corresponds to negatively supercoiled DNA, the top of the arch corresponds to relaxed DNA and the right-hand branch corresponds to positively supercoiled DNA. The upper bands in A and B correspond to form II (open circular) and the middle band in B corresponds to form III (linear SSVl) (pictures courtesy of G. Mirambeau). Panel C shows a one-dimensional agarose gel electrophoresis of SSVl DNA isolated...

Separation between relaxed and nicked DNA is possible using agarose gels containing 2 pg/mL chloroquine (21). 

Estimate by electrophoresis in agarose gel the concentration of DNA verify that the plasmid vector runs as the relaxed (digested) form. 

Figure 10.4. Mobility of linear (+) and relaxed, circular (o) DNA through 0.3 or 0.6% agarose gels, at 0.3 V/cm for 30 or 40 h, respectively, in 0.1 M Tris, 0.09 M boric acid, and 1 mM EDTA.

We now consider relaxation of quadrupolar ions in greater detail. The theory presented applies to any spin- quadrupolar nucleus, though the experimental examples discussed are of 7Rb+ in agarose gels. 

Fig. 3. 87Rb T2s and T2f relaxation times as a function of agarose gel concentration.

Complex relaxation is not however confined to biological tissue. Following a freeze-thaw cycle, non-exponential decay is observed for the water protons in an agarose gel (12) and is also readily observed in meat models made from completely synthetic man-made structures (13) In view of the absence of membranes or any semi-permeable barriers in these wholly fabricated materials, the general relevance of compartmentalisation to the observation of complex relaxation needs to be re-examined. 

C. Results. Agarose - In Figure 1, the effect of one freeze/thaw cycle on the spin-spin relaxation of agarose gel is shown. For the initial homogenous gel, the relaxation can be satisfactorily described by a single exponential process characterised by a T value of 46 m.sec. After freeze/thaw, the decay was complex and required a number of discrete exponentials for its adequate description. The decay was fitted to 3 ... 

Figure 1. Transverse relaxation in 4.8 wt% agarose gels. A homogeneous gel B freeze-damaged...

Fig. 2. Temperature dependence of the 73 maritima DNA gyrase supercoiling activity. Relaxed pBR322 DNA was incubated with the faction contaiiiing active DNA gyrase for 10 min at various temperatures. Samples were analyzed on a 1% agarose gel. Lane— Assay without enzyme.

It should be noted that more than one band will appear on electrophoresis of a plasmid. In general, the covalently closed supercoiled form will comprise most of the DNA, and this runs the most rapidly through an agarose gel because of its compact nature. This is then followed by linear molecules, relaxed (nicked) circular forms and dimer, trimer, and other concatenenes. 

The gelling behaviour of aqueous agarose gels has beat extenavely studied by Nishinari et al. [166,204,520-5293 with various measuring tediniques (stress relaxation, dynamic mechanical behaviour, differential anning calorimetry, optical rotation etc). 

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