Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects.

This report describes the use of a new type of agarose (FastLane agarose) for faster separation of DNA by agarose gel electrophoresis. DNA molecules separated in this agarose exhibited electrophoretic mobilities up to 30% higher than similar separations in standard analytical grade agarose. DNA molecules of all sizes examined showed higher mobilities in FastLane agarose. The mobility increase was predominantly due to the low electroendosmosis of FastLane agarose and was most pronounced in pulsed field gel electrophoresis separations. The magnitude of mobility increase varied depending on the conditions used for electrophoresis.     

Preparation of Agarose with Low Net Negative Charge Density Using an Inexpensive Anion Exchanger

A method of preparing agarose of low net negative charge density by the use of Chromium tanned gelatin is described. Using commercial agarose (agarose c) as standard of comparison the product has a sulphate content approximately 0.1 that of agarose c and its pyruvate content was reduced to zero as shown by NMR in DMSO. The product was still able to adsorb and desorb serum glycoproteins similar to agarose c indicating that charge on the agarose c gel particles played a minor role if any on the binding of the serum glycoproteins.     

Preparation of agarose with low net negative charge density using an inexpensive anion exchanger

A method of preparing agarose of low net negative charge density by the use of Chromium tanned gelatin is described. Using commercial agarose (agarose c) as standard of comparison the product has a sulphate content approximately 0.1 that of agarose c and its pyruvate content was reduced to zero as shown by NMR in DMSO. The product was still able to adsorb and desorb serum glycoproteins similar to agarose c indicating that charge on the agarose c gel particles played a minor role if any on the binding of the serum glycoproteins.     

Enzymatic liquefaction of agarose above the sol–gel transition temperature using a thermostable endo-type β-agarase,Aga16B

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of d-galactose and 3,6-anhydro-l-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol–gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol–gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol–gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40^T, was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol–gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol–gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

     

Recycling agarose

The cost of agarose for DNA gel electrophoresis can be significant in largescale projects. The International Potato Center (CIP) laboratory in Lima-Perú reuses agarose to save money. Due to limited financial resources for research, not uncommon in Latin American laboratories, this practice satisfies a need to control expenses. Recycled agarose can be used for RAPD analysis, or other applications in which no further manipulation of the DNA is necessary. Here we describe an improved CIP method where large amounts of agarose are ultimately equilibrated in water and dried to a powder similar to the original agarose.     

Preparation of electroendosmosis-free agarose gel and exemplification of its use in crossed immunoelectrophoresis

Preparation of electroendosmosis-free agarose is described. The method is based upon the covalent bonding to agarose of a small number of positively charged groups which counterbalance the electroendosmosis-producing effect of the negatively charged groups present in commercial agarose. It is shown how such electroendosmosis-free agarose gel can be combined with advantage with polyacrylamide gels in crossed immunoelectrophoresis.     

Cloning, restriction digestion and DNA labeling of large DNA fragments (greater than or equal to 1 kb) in the presence of remelted SeaPlaque GTG agarose gels.

Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.     

The use of agarose in the determination of anchorage-independent growth

Summary At the present time, growth in agar suspension is one of the best in vitro correlates of tumorigenicity. Growth in agarose, however, has not been evaluated extensively as an in vitro criterion for tumorigenicity. In the present study we have tested 19 cell lines, including six mouse-human hybrids, for growth in agarose and agar in the presence and absence of exogenous hypoxanthine. None of the six nontumorigenic cell lines grew in agar or agarose. Ten of the 13 tumorigenic cell lines grew in both agar and agarose with about equal efficiency. The remaining three tumorigenic cell lines grew well in agarose but poorly or not at all in agar. Hypoxanthine did not stimulate the growth in agar or agarose of any of the cell lines except BHK. We conclude that growth in agarose may be a more sensitive marker for tumorigenicity than growth in agar and that BHK is exceptional in its sensitivity to supplemental purines. This research was supported by DHEW NCI Contract No. NO-CP-2-3234 and by USPHS Medical Scientist Training Grant GM 02042-07.     

Properties of agar: parameters affecting gel-formation and the agarose-iodine reaction

The effects of concentration of agarose, methyl sulphoxide, and substituted agaroses on the mechanism of gel formation have been evaluated with reference to the “Network theory of gel formation”. Factors affecting formation of the agarose gel-iodine color complex were investigated, and the results suggest that the iodine molecules are incorporated between the agarose helices in the Gel II state of agarose.     

琼脂糖凝胶的合适浓度对于回收酶切后质粒载体的重要性

目的：探讨琼脂糖凝胶的不同浓度对回收不同大小的酶切后质粒载体纯度的影响。方法：将2种质粒载体酶切,并经不同浓度的琼脂糖凝胶电泳分析,通过比较酶切前和酶切后载体的相对位置,研究胶浓度对回收酶切后载体纯度的影响。结果：不同浓度的琼脂糖凝胶中,酶切前和酶切后载体的相对位置会发生变化,对能否成功回收到纯度高的酶切后DNA片段有重要影响;质粒大小不同,胶浓度的影响也不同。结论：合适的胶浓度对于回收酶切后质粒载体具有重要意义,应选择合适的胶浓度回收酶切后质粒载体。     

The use of agarose in the determination of anchorage-independent growth.

At the present time, growth in agar suspension is one of the best in vitro correlates of tumorigenicity. Growth in agarose, however, has not been evaluated extensively as an in vitro criterion for tumorigenicity. In the present study we have tested 19 cell lines, including six mouse-human hybrids, for growth in agarose and agar in the presence and absence of exogenous hypoxanthine. None of the six nontumorigenic cell lines grew in agar or agarose. Ten of the 13 tumorigenic cell lines grew in both agar and agarose with about equal efficiency. The remaining three tumorigenic cell lines grew well in agarose but poorly or not at all in agar. Hypoxanthine did not stimulate the growth in agar or agarose of any of the cell lines except BHK. We conclude that growth in agarose may be a more sensitive marker for tumorigenicity than growth in agar and that BHK is exceptional in its sensitivity to supplemental purines.     

PCR特异产物回收纯化方法的比较

方法：采用三种方法对苹果褪绿叶斑病毒RT-PCR的特异DNA产物进行回收纯化。目的：针对不同情况,选择适宜的回收纯化方法。结果：用普通琼脂糖替代低融点琼脂糖,回收纯化后产物的浓度及纯度与低融点琼脂糖法基本一致,完全可以用普通琼脂糖替代低融点琼脂糖进行DNA片段的回收纯化,从而降低成本,简化操作。玻璃奶法的回收纯度明显高于低融点琼脂糖法和普通琼脂糖法,且更快速安全,是采用普通琼脂糖法还是采用玻璃奶法回收纯化DAN片段应以实际需要而定。     

琼脂糖印迹法：观察植物表皮细胞的一种简易方法

本文介绍一种获得完整植物器官表皮细胞大小和数目的简易方法：琼脂糖印迹法。该方法根据琼脂糖凝固时具有可塑性的原理,通过对材料固定、包埋、切胶和显微观察等步骤从而获得材料表皮细胞的轮廓。该方法具有简单迅速、图像清晰、观测结果准确且应用广泛等优点,可使统计植物发育过程中细胞数目及大小的工作变得简单易行。     

Successful preparation and characterization of biotechnological grade agarose from indigenous Gelidium amansii of Taiwan

This paper reports the first successful preparation of biotechnological grade agarose from Gelidium amansii found in Taiwan. The scale-efficiency preparation was achieved by shortening EDTA treatment time through dispersing G. amansii agar in water in the presence of heat and EDTA, removing agaropectin impurity with a heat-compatible and strong-anion exchange resin, and precipitating agarose with a cost-effective isopropanol method. The yield of agarose from prepared G. amansii agar was 11.3%. The acquired agarose has a gel strength of 853 g cm^?2, a sulfate content of 0.14%, a pyruvate content of 1.03%, a degree of electroendosmosis of 0.16 and very limited binding affinity to DNA. The excellent properties of agarose from G. amansii of Taiwan confirm its potential diverse biotechnological applications. This innovative agarose preparation method with the significantly improved scale-efficiency can be modified for large-scale preparation of agarose for use in biotechnological industry and biochemical research.     

Production of cell-enclosing hollow-core agarose microcapsules via jetting in water-immiscible liquid paraffin and formation of embryoid body-like spherical tissues from mouse ES cells enclosed within these microcapsules

We developed agarose microcapsules with a single hollow core templated by alginate microparticles using a jet-technique. We extruded an agarose aqueous solution containing suspended alginate microparticles into a coflowing stream of liquid paraffin and controlled the diameter of the agarose microparticles by changing the flow rate of the liquid paraffin. Subsequent degradation of the inner alginate microparticles using alginate lyase resulted in the hollow-core structure. We successfully obtained agarose microcapsules with 20-50 microm of agarose gel layer thickness and hollow cores ranging in diameter from ca. 50 to 450 microm. Using alginate microparticles of ca. 150 microm in diameter and enclosing feline kidney cells, we were able to create cell-enclosing agarose microcapsules with a hollow core of ca. 150 microm in diameter. The cells in these microcapsules grew much faster than those in alginate microparticles. In addition, we enclosed mouse embryonic stem cells in agarose microcapsules. The embryonic stem cells began to self-aggregate in the core just after encapsulation, and subsequently grew and formed embryoid body-like spherical tissues in the hollow core of the microcapsules. These results show that our novel microcapsule production technique and the resultant microcapsules have potential for tissue engineering, cell therapy and biopharmaceutical applications.     

琼脂糖印迹法: 观察植物表皮细胞的一种简易方法

本文介绍一种获得完整植物器官表皮细胞大小和数目的简易方法: 琼脂糖印迹法。该方法根据琼脂糖凝固时具有可塑性的原理, 通过对材料固定、包埋、切胶和显微观察等步骤从而获得材料表皮细胞的轮廓。该方法具有简单迅速、图像清晰、观测结果准确且应用广泛等优点, 可使统计植物发育过程中细胞数目及大小的工作变得简单易行。     

Electrophoresis, crossed immunoelectrophoresis, and isoelectric focusing in agarose gels with reduced electroendosmotic flow

An ion exchange method using QAE-Sephadex for preparation of agarose with a low electroendosmotic flow and reduced adsorption properties is described. The successful use of such agarose for the separation of highly cationic proteins is illustrated.A method for isoelectric focusing of proteins in gels made from a mixture of purified agarose and a water-soluble non-cross-linked acrylamide polymer is described. This method can be combined with immunochemical identification by electrophoresis of the separated components into antibody-containing agarose gels, also containing such a polymer of acrylamide.     

In vitro cloning of tumor stem cells in semi-solid media containing agar and agarose

Summary The formation of tumor stem cell colonies in vitro has been studied by comparing the growth of three mouse teratocarcinoma derived cell lines and one human teratocarcinoma derived cell line in semi-solid media containing either agar or agarose. We show that agaroses should be used in higher concentrations than agar to obtain comparable results. The maximum number of colonies were obtained in agarose over a broader range of concentrations (1%–4% for SeaPrep and 0.5%–2% for SeaPlaque agarose) than in agar, which allowed anchorage-independent growth of tumor cells only over a narrow concentration range (0.3%–0.5%). Overall, the preparation of media containing agarose was less cumbersome than preparation of agar-containing media, primarily because agaroses gelled more slowly and remained liquid in the physiological temperature range. Furthermore, the transfer of colonies from semi-solid media containing agarose to solid surface tissue culture dishes was much more efficient than the transfer of colonies from agar. The stock solutions of SeaPrep agarose could be kept ready for use for extended periods of time. All these features show that the low melting point agarose has considerable advantages over agar for preparation of semi-solid media for anchorage-independent tumor cell growth.     

Simultaneous use of standard and low-melting agarose for the separation and isolation of DNA by electrophoresis

In this communication we describe the sequential use of standard and low-melting agarose in a single gel slab for the electrophoresis of DNA. This method has the advantages of high resolution and reproducibility characteristic of standard agarose and the ease of manipulation of DNA for direct cloning, sequential digestion and isolation, characteristic of low-melting agarose.