Enzymic methods agarose

Sela, L, Fluorescence of nucleic acids with ethidium bromide an indication of the configurative state of nucleic acids, Biochim. Biophys. Acta 190, 216-219, 1969 Le Pecq, J.B., Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. Methods Biochem. Anal. 20, 41-86, 1971 Borst, P., Ethidium DNA agarose gel electrophoresis how it started, lUBMB Life 57, 145-141, 2005. 

Very recently (84) this specific binding of alloxanthine-like compounds to active xanthine oxidase has been made the basis of a method of separating the active from the inactivated enzyme, using a pyrazolo-(3,4) pyrimidine attached to agarose. 

In a number of cases, e.g. for cloning and DNA sequencing, the DNA must be recovered from the gel in as high a yield as possible and without damage to the molecules. A number of methods have been tried, none entirely satisfactory, but electroelution is most often used. The two major problems with this method are, firstly, that the agarose is often contaminated by sulphated polysaccharides, which are extracted from the gel together with the DNA, and are potent inhibitors of many enzymes and, secondly, that the yield of DNA, particularly for large molecules, is relatively low. 

Alkaliphilic Bacillus sp. AM-001 was aerobically grown to the early stationary phase at 37°C in alkaline medium (pH 9.0) containing 1% konjak mannan. Total chromosomal DNA obtained by the method of Saito and Miura( 14) was digested with Hindlll restriction enzyme. And 2 to 4 kbp DNA fraction of chromosomal DNA was collected by 1% agarose gel electrophoresis. The plasmid pUC19 was digested with Hindlll and then dephosphorylated with bacterial alkaline phosphatase. After the... 

Peptides consisting of residues from GroEL immobilized on agarose have proved effective minichaperones (Altamirano ef al., 1997). The procedure used both column chromatography and batch-wise methods to renature an insoluble protein from an inclusion body, refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Eragments were immobilized by two methods Ni-NTA resin and CNBr-activated Sepharose 4B. 

Two isolation procedures based on methods in category 2 are described in this experiment, a large-scale and a microscale method. Each procedure yields plasmid DNA that is sufficiently pure for size analysis by agarose electrophoresis and for digestion by restriction enzymes as described in Experiment 15. 

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

With the help of this method, His-tagged L-lactate dehydrogenase was immobilized. By pumping pyruvic acid as substrate with NADH as cofactor, it was demonstrated that the enzyme was still active in the microchannel. In this case, cofactor was used up. Srinivasan et al. [433] incorporated PikC hydroxylase from Streptomyces venezuelae into a PDMS-based microfluidic channel with a similar approach. The enzyme was immobilized to Ni-NTA agarose beads with an in situ attachment, following the addition of the beads to the microchannel. This enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin (Scheme 4.104) in about equal amounts with a conversion of >90% at a flow rate of 70nl/min. 

To assay for the presence of supercoiled, circular mtDNA several approaches can be taken. The simplest method is to run a small part of the entire sample (1/20 of the total), either undigested or digested with a restriction enzyme, on a 0.8% (w/v) agarose gel in 1X TBE (Tris-borate-EDTA buffer) and to visualize the DNA by staining the gel with ethidium bromide. If the DNA is undigested by a... 

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