SYBR Staining - Jenn's Method
Remove 10,000x SYBR-Gold and Anti-fade solutions from -20°C freezer.
Keep SYBR in the dark.
Keep SYBR in the dark.
Have 0.02 μm filtered TE, dilution buffer and water on hand enough to make dilutions and wet filters.
Prepare dilutions of sample.
** Mix by inverting tube up and down.
Label frosted slides and get out coverslips.
Cut scotch tape in half for taping down coverslips.
Turn off the lights.
Dilute SYBR 1:10 using 5 μl in 45μl TE.
Put 10 μl drop of Anti-fade onto coverslips.
Label petri dishes:  mark in half and use arrow or number to designate top.
Pipet 2.5 μl diluted SYBR on to top and bottom halves of petri dish (2 drops per dish) and add 97.5 μl TE.
Turn vacuum pump on, but do not turn on vacuum to tower supports.
Pipet water onto top of frosted support.
Place a backing filter on top of tower support and open vacuum to evenly lay down filter.
Turn off vacuum.
Place another drop of water on top of filter and place 25 mm 0.02 μm Anodisc filter on top.
Turn on vacuum.
Clamp tower to support.
Pipet in sample bringing tip very close to filter.
Let filter dry (~30 sec).
With vacuum still running, carefully remove filter and place on drop of SYBR in petri dish.
Stain for 15 min.
Turn off vacuum.
Repeat steps 18 - 23 until all samples are stained.
After staining, place 0.5 ml water on backing filter.
Pick up stained filter with forceps and place on top of water droplet.
Turn on vacuum for ~30 sec to dry filter.
Remove filter with vacuum still on and place coverslip with anti-fade on top.
Set down forceps and place filter and coverslip onto slide.
Tape down one end.
Put 2 coverslips per slide and tape in the middle to hold down coverslips.
Examine immediately or place in freezer for 1 hr to improve staining.
