Next, 1 µl of each product was used in a touchdown PCR reaction w

Next, 1 µl of each product was used in a touchdown PCR reaction with primers 338f-518R with a profile of 5 min at 95 °C, 10 cycles of 30 s at 95 °C, 45 s at (60 °C – 0.5 °C), 1 min 30 MI-503 s at 72 °C, 13 cycles of 30 s at 95 °C, 45 s at 55 °C, 1 min 30 s at 72 °C and a final elongation step of 65 min at 72 °C. This PCR-DGGE provided a similar profile as the non-nested PCR-DGGE, but the eukaryotic 18S rRNA gene was absent. The empty lane of the no-template control indicated the absence of contamination. The Bio-Rad DCode system was used for the analysis. Gels with 8 % (w/v) polyacrylamide

were ran in 1 x TAE (40 mM Tris-Cl, 20 mM glacial acetic acid, 1 mM disodium

EDTA.2H2O, pH 7.4) with a denaturing gradient of 45 to 60 % (100 % denaturant contains 7 M urea and 40% formamide) for 16 h at 38 V. Gels were stained with SYBR-Green and visualized under UV light (Isogen ProXima 16 Phi system, Isogen Life Science, Sint-Pieters-Leeuw, Belgium). To analyze the different bands of the DGGE-pattern, bands were excised from gel, and washed for three times in sterile water. DNA was then eluted from the gel by heating at 37 °C with 100 µl of sterile water; 1 µl was used for reamplification. PCR-products were cloned in the pGEM-T vector, reamplified using primer pair 338F-518R and run check details on a PCR-DGGE gel to discriminate the different bands. Plasmids corresponding to bands of interest were sent to LGC genomics for sequencing. Fluorescence in situ hybridisation The co-localization of Rickettsia and Wolbachia in the reproductive tissues was confirmed with a fluorescent in situ hybridization (FISH). The analysis was carried out following the protocol of Crotti et al. [45] Tacrolimus (FK506) for whole-mounted samples with slight modifications. Ovaries of infected and cured M. pygmaeus females were collected in a drop of 1 x PBS under a stereomicroscope, fixed for 1 h in 4 % paraformaldehyde in 1 x PBS and washed three times with

1 x PBS. The ovaries were then incubated for 1 min in a 100 µg/ml pepsin solution and washed again three times with 1 x PBS and one time with the hybridization buffer without probe (2 x SSC, 50 % formamide). Hybridization was carried out overnight at 46°C in hybridization buffer with 10 pmol/ml fluorescent probe. The next day, samples were washed in hybridization buffer without probe, two times in 0.1 x SSC and two times in 1 x PBS. Subsequently, the samples were whole-mounted with Vectashield Mounting Medium (Vector Labs, Burlingame, CA, USA) and images were acquired using a Nikon A1R confocal microscope, mounted on a Nikon Ti body, using a 60 x (NA1.4) oil objective.

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