SYBR green staining 1.doc Pagina 1 van 2 SYBR Green staining of cells Use sterilised solutions, filter sterilise solutions before use (0.2 um) filter, to get rid of possible contaminating cells (don’t do that with the SYBR green, it will absorb to the filter) SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to double-stranded DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 498 nm) and emits green light (λmax = 522 nm). Fixation + storage Water samples: - Add 100 ul 37% formaldehyde per 1 ml sample. Incubate o/n at 4oC - Centrifuge at high speed for 10 minutes, remove supernatant. - Wash pellet once with PBS (1 ml), centrifuge and then dissolve pellet in 1 ml PBS, continue directly with staining. - For long term storage: use 0.5 ml 2xPBS + 0.5 ml ethanol instead of 1 ml PBS Sediment samples - Mix 2 g of sample with 6 ml PBS and 0.6 ml 37% formaldehyde. Incubate o/n 4oC - Continue as for water samples. Extraction of cells from sediment samples - centrifuge samples, at low speed 2’ 1000 rpm - remove supernatant and add 6 ml of 0.1% sodium pyrophosphate (NaPP) - vortex 4 x 30 sec - centrifuge at low speed 2’ 1000 rpm, collect supernatant and add fresh 0.1% NaPP - repeat previous 3 steps, 3 times. - Combine all the supernatants, centrifuge at high speed (15’ 20000 rpm) and dissolve in 6 ml PBS or PBS-50% ethanol. SYBR green staining 1.doc Pagina 2 van 2 Staining - Add 50 ul of 1/100 diluted SYBR Green I in PBS to 1 ml of (appropriately diluted) sample - Incubate for 30 minutes in the dark - Attach filter (isopore membrane filters, 0.2 um GTBP (polycarbonate) from Millipore) to filtration funnel, don’t use too high pressure - Wash filter once with 2 ml PBS - When the filter is dry, add the sample to the filter - Wash with 2 x 2 ml PBS - Remove filter and put on microscope slide, immobilise the filter by putting the filter on top of a part of the slide which has been smeared with immersion oil - Add a drop of non-fluorescent immersion oil on top of the filter. View the slide under the microscope, under blue light. - Use program GFP_mcf at the cofocal microscoop E. coli cells .