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Title: Electro-hydrodynamic extraction of DNA from mixtures of DNA and bovine serum albumin
We report separation of genomic DNA (48 kbp) from bovine serum albumin (BSA) by the electro-hydrodynamic coupling between a pressure-driven flow and a parallel electric field. Electro-hydrodynamic extraction exploits this coupling to trap DNA molecules at the entrance of a microfluidic contraction channel, while allowing proteins and salts to be flushed from the device. Samples (10 μL) containing λ-DNA (1 ng) and BSA (0.3 mg) were injected directly into the device and convected to the contraction channel entrance by a flowing buffer solution. The DNA remains trapped in this region essentially indefinitely, while proteins and salts are eluted. The effectiveness of the concept has been assessed by fluorescence measurements of DNA and BSA concentrations. Electro-hydrodynamic extraction in a single-stage device was found to enhance the concentration of DNA 40-fold, while reducing the BSA concentration by four orders of magnitude. The relative concentrations of DNA to BSA at the contraction channel entrance can be as large as 1.5 : 1, corresponding to an A260/280 ratio of 1.9. The maximum yield of DNA from a salt-free solution is 50%, while salted (150 mM) solutions have a lower yield (38%).  more » « less
Award ID(s):
Author(s) / Creator(s):
; ; ;
Date Published:
Journal Name:
The Analyst
Page Range / eLocation ID:
5532 to 5538
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Key points

     Accumulation of inorganic phosphate (Pi) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Piinto SR are fully established.

     We showed that Pipromoted Ca2+release at concentrations below 10 mmand decreased it at higher concentrations. This decrease correlated well with that of [Ca2+]SR.

     Pre‐treatment of permeabilized myofibres with 2 mmClchannel blocker 9‐anthracenecarboxylic acid (9AC) inhibited both effects of Pi.

     The biphasic dependence of Ca2+release on [Pi] is explained by a direct effect of Piacting on the SR Ca2+release channel, combined with the intra‐SR precipitation of Ca2+salts. The effects of 9AC demonstrate that Pienters the SR via a Clpathway of an as‐yet‐undefined molecular nature.


    Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi). Pidiffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+salts, thus contributing to the impairment of Ca2+release. Information on the Pientrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+spark frequency is correlated with the Ca2+load of the SR and can be used to monitor this variable. We studied the effects of Pion Ca2+sparks in permeabilized fibres of the frog. Relative event frequency (f/fref) rose with increasing [Pi], reaching 2.54 ± 1.6 at 5 mm,and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi] = 80 mm. Measurement of [Ca2+]SRconfirmed a decrease correlated with spark frequency at high [Pi]. A large [Ca2+]SRsurge was observed upon Piremoval. Anion channels are a putative path for Piinto the SR. We tested the effect of the chloride channel blocker 9‐anthracenecarboxylic acid (9AC) on Pientrance. 9AC (400 µm)applied to the cytoplasm produced a non‐significant increase in spark frequency and reduced the Pieffects on this parameter. Fibre treatment with 2 mm9AC in the presence of high cytoplasmic Mg2+suppressed the effects of Pion [Ca2+]SRand spark frequency up to 55 mm[Pi]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Piimpairs Ca2+release by accumulating and precipitating with Ca2+inside the SR, thus contributing to myogenic fatigue.

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  2. Abstract

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