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 .
