Prestaining DNA with SYBR Green I.

Paul St. Amand PST at KSU.KSU.Edu
Tue Aug 29 14:53:41 EST 1995


There seems to be a lot of interest on using SYBR Green I. This is how I
use it.  If anyone improves on this protocol, please post! (I am in no way
associated with Molecular Probes.)

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Prestaining DNA with SYBR Green I prior to running on agarose gels.

Solutions needed:

10X loading buffer:

Heat 100ml water, stir rapidly, slowly add 25g Ficol (will take 5-10
minutes to dissolve), add 0.05g Bromophenol Blue, add 0.05g Xylene Cyanole
FF, continue stirring until well mixed (about 30 minutes).

SYBR Green I:

Purchase from Molecular Probes [U.S. phone # (503) 465-8300] Comes as a
10,000X concentrate. Must be stored without light, desiccated, and at
-20°C. Current cost is U.S. $250.00 per ml (about U.S. $0.25 per 96 well
plate using the following protocol).

Loading buffer with SYBR Green:

To a 1600ul aliquot of 10X loading buffer, add 2ul of 10,000X SYBR Green,
vortex. Must be stored without light and at -20°C. Add 6 to 8ul of this
solution per 50ul PCR (1600ul is enough for two 96 well plates at 8ul per
50ul reaction and the final concentration of SYBR Green is about 1.7X).


1. Thaw loading buffer/SYBR Green in 65°C bath, but don't leave in more
than 5 minutes.

2. Vortex and then centrifuge loading buffer/SYBR Green.

3. Add a uniform amount of loading buffer/SYBR Green to each PCR.

4. Heat PCR samples to 70°C for 1-2 minutes (this will help get the drop
of buffer through the mineral oil overlay).

5. Let PCR samples stand at room temperature for 10-15 minutes.

6. Mix sample very well with pipette prior to loading on agarose gel.

Tips: SYBR Green causes bands to move somewhat more slowly than Ethidium
Bromide. Cover the gel with as little running buffer as possible (less
than lmm deep). Always mix sample in the pipette by pumping up and down
several times. Refreeze unused loading buffer/SYBR Green right away.
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Paul C. St. Amand    (PST at KSU.KSU.Edu)
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