How to dilute SYBR green and
ROX for real time PCR
Molecular
Probes SYBR green product:
Sigma's
SYBR green product: S9430
Molecular
Probes Rox product: 6-carboxy-X-rhodamine
Sigma's
ROX product:
Molecular
Probes SYBR green product:
Sigma's
SYBR green product: S9430
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Delivered as 0.5ml 10 000X concentration.
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Store as 2µl aliquots at -70°C (temperature: see ref).
Total number of aliquots: 250.
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Prepare intermediate solution of 50X with H2O.
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Store as aliquots at -20°C. Diluting 2µl 200 times results
in 400µl of 50X SYBR , sufficient for more than 5000 25µl reactions.
Choose aliquot volume depending on estimated usage. Keep up to one week
at 2-8°C
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For working solution 1:150 000, dilute 50X to 3.75X: take 30µl
of 50X and dilute to 400µl. working solution aliquots should be discarded
after use.Use working solution to reach final concentration of 0.075X
in reaction; To a 25µl reaction volume add 0.5µl of 3.75X
SYBR green.
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For working solution 1:100 000, dilute 50X to 5X: take 100µl
of 50X and dilute to 1ml. Use working solution to reach final concentration
of 0.1X in reaction; To a 25µl reaction
volume add 0.5µl of 5X SYBR green.
Reference: Yin
et al 2001. Note: our dilution differs from theirs! they use final
concentration of 0.5X in reaction.
SYBR dilution test: Run 18S (product
415bp) and GAPDH (product 255bp) primer pairs on 30ng template (muscle
cDNA from RT) in the following final concentrations of SYBR green (Run
triplicates; less than 2µl needed of all SYBR concentrations!); concentrations
to the left are the ones desired in the tube.
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1X (1:10 000) Dilute Stock aliquot to 100X: Take 2µl
stock dilute to 200µl.
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0.5X (1:20 000) Take 100µl of 1, dilute to 200µl
(50X working solution)
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0.25X (1:40 000) Take 50µl of 2, dilute to 100µl
(25X working solution)
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0.125X (1:80 000) Take 50µl of 3, dilute to 100µl
(12.5X working solution)
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0.0625X (1:160 000) Take 50µl of 4, dilute to 100µl
(6.25X working solution)
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0.03125X (1:320 000) Take 50µl of 5, dilute to
100µl (3.125X working solution)
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1X (1:10 000) See above
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0.1X (1:100 000) Take 10µl of 1, dilute to 100µl
(10X working solution)
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0.075X (1:150 000) Take 5µl of 2, dilute to 7.5
(7.5X working solution)
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0.05X (1:200 000) Take 50µl of 2, dilute to 50µl
(5X working solution)
Test performed 020510,13,14: result: SYBR seems to inhibit the PCR-reaction
even at low concentrations. The most diluted SYBR resulted in the lowest
CT-values (earliest detection of product). However, the plataeu
was reached at correspondingly lower fluorescence intensity values for
low concentration of SYBR. SYBR concentration of 0.075X in reaction was
found to be a functional tradeoff.
Molecular
Probes Rox product: 6-carboxy-X-rhodamine
Dissolve 15mg of ROX in 0.5ml DMSO (Sigma) and dilute to
600nM with H2O.
Store at -20°C
Sigma's ROX product:
Suggestions and Comments