ii)Įdėjus 1 ml 0,1 M fosfatinio buferinio tirpalo (FB; pH 7,0) ir 5 lašus 1N HCl, nustatomas pH 7,0. Patikrinti pH indikatorinėmis juostelėmis ir nustatyti pH, pridedant HCl arba NaOH. (DĖMESIO! NENAUDOTI PH MATAVIMO PRIETAISO, JEI NAUDOJAMI PARAFORMALDEHIDO TURINČIUOSE TIRPALUOSE) iii) Kol bus vėliau naudojamas, tirpalą filtruoti 0,22 mM membraniniu filtru ir saugoti nuo dulkių 4 °C temperatūroje.
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3.3X Hybmix tirpalas NaCl 2,7 M Tris-HCl 60 mM(ph 7,4) EDTA (filtruotas steriliu filtru ir sterilizuotas autoklave) 15 mM Kaip reikalaujama, praskiesti 1 kartą.
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4.Hibridizavimo darbinis mišinys 1 kartą koncentruotas Hybmix Natrio dodecilsulfatas (SDS) 0,01 % Formamidas 30 % Bandinys EUB 338 5 ng/ml Bandiniai OLI-1 arba OLI-2 5 ng/ml Pagal 1 lentelėje pateikiamus apskaičiavimus paruošti hibridizavimo mišinio kiekius. Kiekvienos plokštelės (į kurios duobutes įpilama po 2 skirtingus bandinius su pakartojimais) reikia 90 ml hibridizavimo mišinio. (DĖMESIO! FORMAMIDAS LABAI NUODINGAS, TODĖL MŪVĖKITE PIRŠTINES IR IMKITĖS ATITINKAMŲ SAUGUMO PRIEMONIŲ!) 1 lentelė. Siūlomi kiekiai hibridizavimo mišiniui paruošti Plokštelių skaičius 1 4 6 8 10 Sterilus YGV 23,1 92,4 138,6 184,8 231,0 3x hybmix tirpalas 300,0 120,0 180,0 240,0 300,0 1 %SDS 0,9 3,6 5,4 7,2 9,0 Formamidas 27,0 108,0 162,0 216,0 270,0 Mėginys EUB 338 (100 ng/ml) 4,5 18,0 27,0 36,0 45,0 Mėginys OLI-1 arba OLI-2 (100 ng/ml) 4,5 18,0 27,0 36,0 45,0 Bendras tūris (ml) 90,0 360,0 540,0 720,0 900,0 Pastaba. Visus tirpalus, turinčius šviesai jautrių oligonukleotidinių bandinių, saugoti tamsoje -20 °C temperatūroje. Naudojimo metu saugoti nuo tiesioginės saulės šviesos ar elektros šviesos.
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5.0,1 M fosfatinis buferinis tirpalas, pH 7,0 Na2HPO4 8,52 g KH2PO4 5,44 g Distiliuotas 1,00 l vanduo Ingredientai ištirpinami, patikrinamas pH, ir tirpalas 15 minučių sterilizuojamas autoklave 121 °C temperatūroje. ______________ 8 priedėlis Baklažano ir pomidoro kultūra Pomidoro (Lycopersicon esculentum) arba baklažano (Solanum melongena) sėklos pasodinamos pasterizuotame sėklų komposte. Daigai su visiškai išsiskleidusiomis sėklaskiltėmis (10–14 dienų) persodinami į vazonėlį su pasterizuotu kompostu.
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Pomidorai arba baklažanai turi būti auginami šiltnamyje esant tokioms aplinkos sąlygoms: dienos ilgumas: 14 valandų arba natūralus dienos ilgumas, jeigu diena ilgesnė; temperatūra: dieną nuo 21 iki 24 °C, naktį nuo 14 iki 18 °C.
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Jautri užkrėtimui pomidorų veislė:„Moneymaker“ Jautri užkrėtimui baklažanų veislė:„Black Beauty“ Tiekėjas: žr. svetainę http://forum. europa. eu. int/Public/irc/sanco/Home/main ______________ NUORODOS 1. Amann, R. I., L. Krumholz and D. A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J. Bacteriol. 172: 762–770.
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27.Taghavi, M., Hayward, A. C., Sly, L. I., Fegan, M. 1996. Analysiss of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the blood disease bacterium of banana based on 16S rRNA gene sequences. International Journal of Systematic Bacteriology 46; 10–15.
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28.Van Der Wolf, J. M., Bonants, P. J. M., Smith, J. J., Hagenaar, M., Nijhuis, E., Van Beckhoven, J. R. C., Saddler, G. S., Trigalet, A., Feuillade, R. 1998. Genetic diversity of Ralstonia solanacearum Race 3 in Western Europe as determined by AFLP, RC-PFGE and rep-PCR. In: Prior, P., Allen, C and Elphinstone, J. (eds.) Bacterial wilt disease: Molecular and Ecological Aspects. Springer (Berlin) pp. 44–49.
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29.Weiler, S. A., Elphinstone, J. G., Smith, N., Stead, D. E. and Boonham, N. 1999. Detection of Ralstonia solanacearum strains using an automated and quantitative flourogenic 5' nuclease TaqMan assay. Applied and Environmental Microbiology 66; 2853–2858.
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