Sybr Green I in real-time PCR reaction (View forum version)



Michaelro

Posted 13 October 2009 - 11:54 PM

Hi everyone
We want to add manually Sybr Green I dye into the real-time PCR reaction
We intend to use S9430 product from Sigma which is 10,000X solution of Sybr Green I in DMSO
Does anybody have experience with this product or similar products in real-time PCR?
Thanks for your help
Michael

fhsantanna

Posted 19 October 2009 - 10:50 AM

Yes, it functions perfectly.
I use the SyBR Green 10000x of Invitrogen. I dilute 100X in DMSO (diluted stock final concentration: 100X) and when I am going to perform the PCR reaction I dilute this diluted SYBR stock a hundred more times in water (1X). If you are going to perform a 20 uL reaction, you have to put 2 uL of SYBR Green 1x, so, the final concentration of SYBR in the real-time PCR reaction will be 0,1X.

Prep!

Posted 23 October 2009 - 07:12 PM

well fhsantanna is absolutely right!!!
but frankly i have never heard of a 10000X conc SYBR dye.. or may be its just the dye and not the master mix which is generally available with Roche or Eurogentec or ABI which contails Mg+2 ions and dNTPs and TAq polymerase.

Michaelro

Posted 30 October 2009 - 03:47 AM

Yes, it functions perfectly.
I use the SyBR Green 10000x of Invitrogen. I dilute 100X in DMSO (diluted stock final concentration: 100X) and when I am going to perform the PCR reaction I dilute this diluted SYBR stock a hundred more times in water (1X). If you are going to perform a 20 uL reaction, you have to put 2 uL of SYBR Green 1x, so, the final concentration of SYBR in the real-time PCR reaction will be 0,1X.


Thank You very much
It was very helpful
Some guys in our lab have a hard time with the Syber ready mixes so I advised them to introduce the syber green manually into reaction with the DNA polymerase that works well.
Best
Michael