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


    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.
 

  1. 1X (1:10 000)    Dilute Stock aliquot to 100X: Take 2µl stock dilute to 200µl.
  2. 0.5X (1:20 000)    Take 100µl of 1, dilute to 200µl (50X working solution)
  3. 0.25X (1:40 000)    Take 50µl of 2, dilute to 100µl (25X working solution)
  4. 0.125X (1:80 000)    Take 50µl of 3, dilute to 100µl (12.5X working solution)
  5. 0.0625X (1:160 000)    Take 50µl of 4, dilute to 100µl (6.25X working solution)
  6. 0.03125X (1:320 000)    Take 50µl of 5, dilute to 100µl (3.125X working solution)
  1. 1X (1:10 000)    See above
  2. 0.1X (1:100 000)    Take 10µl of 1, dilute to 100µl (10X working solution)
  3. 0.075X (1:150 000)    Take 5µl of 2, dilute to 7.5 (7.5X working solution)
  4. 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:



 
 

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