Jan 07, 2016
SYBR Gold Staining for viral enumeration using 13 mm Anodisc 0.02 ?m filters
- Li Deng & Jennifer Brum1
- 1Matthew Sullivan Lab, University of Arizona/Ohio State University
- VERVE Net
- Sullivan Lab
Protocol Citation: Li Deng & Jennifer Brum 2016. SYBR Gold Staining for viral enumeration using 13 mm Anodisc 0.02 ?m filters. protocols.io https://dx.doi.org/10.17504/protocols.io.c7cziv
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 24, 2015
Last Modified: November 09, 2017
Protocol Integer ID: 964
Preparation of filter holders
Preparation of filter holders
Cut a 3 ml syringe as a filter funnel:
(Fig 1)
Take a gasket from 13 mm MILLIPORE filter holder (FISHER, SX0001300).
Fig 2
Obtain a clamp for 25 filter funnel.
Make dilution of virus prep in 0.02um filtered seawater to a concentration of ~E+07 particles ml-1.
Prepare working solutions of SYBR-Gold
Prepare working solutions of SYBR-Gold
Thaw the commercial stock of SYBR-Gold in the dark at RT and centrifuge at ~3000 rpm for 5 minutes because SYBR-Gold is in DMSO.
00:05:00
Centrifuge at ~3000 rpm for 5 minutes.
00:05:00
Note
SYBR-Gold is in DMSO.
Dilute SYBR-Gold in 0.02 µm filtered TE buffer to 100x (10 µl in 990 µl TE buffer).
Note
This working stock can be stored at -20°C in small aliquots and re-thaw one time.
Add 1 µl of SYBR working stock in 49 µl 0.02 µm filtered TE buffer in a plastic Petri dish.
Note
Can have 4 drops for staining 4 filters in one dish.
Cover the dish by aluminum foil.
Note
SYBR is light sensitive.
Set up the filtration unit, connecting it to a vacuum.
Note
Set up the vacuum no higher than 5 mm Hg.
Add a few drops of 0.02 µm filtered mQ on the filter base and place a 25 mm 0.2 nitrocellulose filter (the support filter) on top of the water.
Fig 3
Switch on the vacuum, the support filter should be flat on the filter base.
Note
This support filter is good for several samples as long as it remains flat and no air bubbles between filter and base.
Place a 13 mm Anodisc filter on the wet nitrocellulose filter.
Fig 4
Preparation of filter holders
Preparation of filter holders
Place a gasket on the 13 mm Anodisc filter.
Fig 5
Place the syringe filter funnel carefully on the gasket and apply the clamp.
Fig 6
Switch on the vacuum and add samples for filtration, leave the vacuum on for 1 more minutes after sample drained completely.
Leave the vacuum on for 1 more minutes after sample drained completely.
00:01:00
Take away the clamp and syringe filter funnel while vacuum is on.
Carefully push the filer to the edge of the filter base by tweezers while vacuum is on to remove the filter.
Fig 7
Dry filter membrane on Kimwipes in the dark at RT completely.
Note
Better in a paper box.
Remove membrane and place viruses-side up on staining solution in the Petri dish for 15 min, cover the Petri dish by aluminum foil.
00:15:00
Cover the Petri dish by aluminum foil.
Dry filter membrane again on Kimwipes in the dark at RT completely.
Note
Better in a paper box.
Pipet 10 µl antifade solution on a microscope slide and place the stained filter membrane on top of it. Pipet 10 µl antifade solution on a coverslid and carefully place it on the filter to avoid bubbles.
Pipet 10 µl antifade solution on a coverslid and carefully place it on the filter to avoid bubbles.
Place slide at –20°C to enhance fluorescence.
Read slides using 100x oil immersion objective and inverted fluorescent microscope.