Relaxation agarose

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. 2. (A) 87Rb triple-quantum filtered NMR spectrum of 2g% agarose in RbCl. (B) Dependence of the triple-quantum signal amplitude on the triple-quantum creation time—a biexponential fit to the peak heights of the series of spectra yields the and T2f relaxation times. From Ref. 51, with permission.

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

Fig. 6. The fast relaxation rate, R2f, of 87Rb+ in agarose. The form of the line fitted to the first 4 points is R2p = 834( 359) + [agarose] x 976( 67). The last point was omitted as it did not fall within the linear region of the two-site exchange model.

The experimental dependence of Rx for 87Rb+ as a function of agarose concentration and magnetic field strength is shown in Fig. 7, and the theoretical interpretation of these results is very similar to that used for the transverse relaxation case. 

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. 

B. Materials. Agarose was from Marine Colloids, Inc. (REX5468), and used without further purification. A sample of gel was prepared by pressure cooking a 4.8 wt% dispersion of agarose in distilled water for 10 min. Approximately 0.3 ml of the solution was placed in a 8 mm O.D. NMR tube and gelled by cooling. After relaxation measurements on the gel had been performed, the sample was frozen rapidly in liquid nitrogen, thawed and remeasured. 

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...

Agarose. In previous work (1 ) it has been shown that the observed relaxation in homogeneous gels is described by the fast exchange approximation. 

---