Do you know the white-colored natural powder on my own plaisant

Do you know the white-colored natural powder on my own plaisant

m. adansonii.

The library was screened by colony hybridization applying nick-translated genomic AHB DNA. Particular person colonies exhibiting the finest hybridization intensities ended up picked for even more analyses. Isolated clones from such colonies possible represented very repetitive DNA sequences. Estimates recommend that ten% to 11% of the honey bee genome is comprised of sequences recurring one hundred or extra periods.

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Crain, et al. (1976), Chromosoma, Vol. A. and R. W.

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Brosemer (1974) J. Insect Physiol. , Vol. c. Library Construction. A DNA library was manufactured making use of common procedures (Maniatis, et al. , Molecular Cloning: a Laboratory Guide, Cold Spring Harbor Laboratory, (1982)).

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Genomic DNA attained from thirty adult employee bees was digested with the restriction endonuclease Sau3A. Plasmid pUC9 was digested with Bam Hello, treated with bacterial alkaline phosphatase, phenol/chloroform extracted, and ethanol precipitated. Honey bee DNA and plasmid ended up incubated with each other and ligated with T4 DNA Ligase.

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The plasmids ended up then remodeled into Escherichia coli (E. coli), pressure TBl, which had been produced skilled by the CaCl 2 approach. Recombinants that contains honey bee DNA inserts had been determined by a absence of B-galactosidase activity in the presence of Xgal (4-Bromo-four-chloro-3-indolyl-B-D-galactopyranoside). d.

Identification of Repetitive Elements. The genomic library was screened by colony hybridization to detect clones containing repetitive DNA. Recombinant clones have been grown on agar plates made up of )-deoxycytidine by nick translation employing a commercially accessible kit (Bethesda Investigate Laboratories) to a precise action of 1-3×10 eight cpm. Nitrocellulose filters were being prehybridized in a typical prehybridization alternative (Maniatis, et al. , supra. ) containing a hundred microgram/ml of yeast RNA for hour at 37° C. , adopted by hybridization with the heat-denatured 32 P-labeled probe in the similar solution for eighteen hrs at 42° C.

Filters were being washed in 2X SSC (Regular saline citrate) as soon as at space temperature for 2 minutes and the moment at 42° C. for 20 minutes, and then two times at 20 minutes every single in . 3X SSC at 68° C. Autoradiography was carried out at -70° C. for 18 hrs working with an intensifying display. This hybridization treatment and circumstances were being applied commonly in other components of the course of action under, as indicated. e. Southern Blots. Genomic DNA, independently extracted from five to ten adult worker bees from personal colonies, was digested with many restriction endonucleases together with: Eco RI, Sau3A, Hae III, Hinf I, Hpa II, Alu I, Hind III, Bam Hi, and Pvu II. The restriction fragments were being size-fractionated on . 6 to one. % agarose gels.

The DNA was transferred to nitrocellulose filters by the Southern blotting procedure (Southern, supra. ). The filters had been baked and prehybridized as described previously.

Honey bee DNA inserts had been isolated from recombinant plasmid by double-digestion with Eco RI and Hind III, followed by electrophoresis in a one. to one. 5% very low melting temperature (LMT) agarose gel.