I use StepOnePlus Real-Time PCR System for relative gene expression studies. I follow SYBR Green chemistry. Is it advisable to use 0.5X SYBR Green Master Mix instead of 1X?
Thanks in advance.
We tried it (0.5x SYBR) successfully with ABI system. But for Agilent system 0.75x SYBR gives flawless result than 0.5x SYBR.
We always use 0,5x SYBR instead of 1x. It works with our system (Biorad CFX96 Touch™ Real-Time PCR Detection System)
We tried it (0.5x SYBR) successfully with ABI system. But for Agilent system 0.75x SYBR gives flawless result than 0.5x SYBR.
I do not concur -- in case your SYBR master mix includes mono- and divalent salts (Na+ and Mg2+), which is the case for most ready-to-use master mixes, and you are changing its concentration to 0.5x, you would change your reaction chemistry drastically.
A lower concentration of those salts can lead to an unknown change in Tm and subsequent unspecific binding. Take a look at your melting curve first derivatives. They should peak high and only once. If they don't, you have a problem. Frankly, I would never change the concentration of master mixes, especially if you do not know what's in it.
I agree with Felix. were it just SYBR dye a lower concentration would be fine, but for a ready master mix I really would not recommend diluting it, or you could risk compromising your assay.
I agree with Felix and Enos. Instead of reducing the SYBR concentration, you can try reducing the total reaction volume, thereby increasing the total number of reactions. To the best of my knowledge, StepOne can do reactions with as low as 10 uL volume.
SD
That's what I thought he meant. Reducing the overall reaction volume.
Thank you all for your kind and valuable suggestions.
With regards.
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