![]() Incubate at 68 ☌ for 1hr to pre-hybridize.ĭuring pre-hybridization, start the probe transcription reaction. Put cross-linked nylon membrane in the hybridization bottle with the RNA-side up.Īdd 10 ml hybridization buffer (for small hybridization bottle) to the membrane. Nonspecifically bound probes are washed away after hybridization. The probes are then hybridized to the membrane. Use in vitro T7 transcription to make radioactively labeled RNA probes complementary to RNA transcript of interest. Gel can be illuminated with UV to check whether there is any remaining RNA. UV crosslink the membrane twice to fix the RNA on the membrane and use a fine marker to mark the edge of the side with RNA.Ĭlean the gel transfer system thoroughly by rinsing with plenty of H 2O.Īfter the gel transfer, the gel area inside the window of green gasket should be half as thick as the gel outside the window. Dry the nylon membrane between two sheets of filter paper. Remove the gel and take out the nylon membrane. When the transfer is over, remove the sealing frame and drain the buffer. Occasionally check the buffer level to make sure it is above the gel. Place the lid on and transfer for 90 mins at 5 inches of Hg. Gently pour 1 L of 10× SSC buffer into the reservoir. Press the gel and along the window gently to apply extra pressure to help the vacuum sealing. Start the vacuum source and adjust the pressure to 5 inches of Hg. Place the sealing frame on top of the vacuum stage and lock it. Remove all the air bubbles between gel and the nylon membrane. Also make sure the gel overlaps with the gasket by at least 5 mm. Gently place the gel on top of the gasket with the well-side up. Make sure the gasket covers the seal o-ring while the membrane/filter paper overlaps with the window of the gasket. Place the plastic gasket on top of the membrane/filter paper. Wet the seal o-ring on the base unit with H 2O. Make sure there is no air bubble between membrane and filter. Place the wetted nylon membrane on top of the filter paper. Put wet filter paper on the vacuum porous stage and make sure the filter paper is in the area where the cut window of the green plastic gasket is going to be. Wet the nylon membrane and filter paper first in H 2O and then 10× SSC buffer Cut a filter paper with the same size as the nylon membrane.įill the wells of the RNA gel with melted agarose. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.RNA is transferred from gel to nylon membrane using vacuum gel transfer system.Ĭut a nylon membrane about (or bigger than) the size of the denaturing RNA gel. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers compared to strains not forming biofilms. The icaA gene was partly sequenced for 22 food related strains from nine different species of Staphylococcus, and their icaA genes were found to have a DNA similarity of 69-100% to previously sequenced icaA genes. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food related strains tested. The biofilm forming strains belonged to nine different coagulase negative species of Staphylococcus. Glucose and sodium chloride stimulated biofilm formation. Seven and 21 of 144 food related strains were found to be strong and weak biofilm formers, respectively. In the present work staphylococci from the food industry were found to vary widely in their ability to form biofilm on polystyrene. Biofilm formation may also be of importance for survival and virulence in food related staphylococci. In clinical staphylococci presence of the ica genes and biofilm formation are considered important for virulence.
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