Master Mix for Real-Time PCR (TaqMan)

[Leowe]

Hey guys!
First, i want to tell you sorry for my bad english
there is my problem:
So, i need to make Master Mix =)
what a differense from basic PCR? I mean...eeemmm...Do anybody speak Russion?
Eh.. Do you know any links for literature about Master Mix for Real-Time PCR?
Smth like: Total concentration for reagents (total volume 50 ml):
DNA ~ 100-400 ng per reaction
Tag ~ 1 unit/ml ~ 1 ml per reaction
Primer A, Primer B ~ 1 ml per reaction (0.2 pMol) 10pMol/ml in Stock
Probe ~
MgCl2 ~ 2-2.5 mM per reaction
dNTP ~ 400 mM per reaction
Rox ~

Do i need Rox for detect Luminescence? What concentration of Probe do i need for reaction?

Unfortunately all of protocols ive seen, autors just wrote "WasterMix" without detalisation.

10x =)

[molecule2005]

Hey there Leowe!


A real-time PCR reaction mixture can be either 50 ml or 25 ml. Prepare the following mixture in each optical tube.
25 ml SYBR Green Mix (2x)
0.5 ml liver cDNA
2 ml primer pair mix (5 pmol/ml each primer)
22.5 ml H2O OR 12.5 ml SYBR Green Mix (2x)
0.2 ml liver cDNA
1 ml primer pair mix (5 pmol/ml each primer)
11.3 ml H2O


http://pga.mgh.harvard.edu/primerbank/PCR_protocol.html

Also

In addition to the use of ROX, a master mix should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility (ROX will be within the master mix).

http://www.dorak.info/genetics/realtime.html

Please let us know your successes and failures!
Hope you get many success.

[Leowe]

Originally Posted by molecule2005

Hey there Leowe!


A real-time PCR reaction mixture can be either 50 ml or 25 ml. Prepare the following mixture in each optical tube.
25 ml SYBR Green Mix (2x)
0.5 ml liver cDNA
2 ml primer pair mix (5 pmol/ml each primer)
22.5 ml H2O OR 12.5 ml SYBR Green Mix (2x)
0.2 ml liver cDNA
1 ml primer pair mix (5 pmol/ml each primer)
11.3 ml H2O



Also

In addition to the use of ROX, a master mix should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility (ROX will be within the master mix).


Please let us know your successes and failures!
Hope you get many success.

Thx for reply!
Is there any differense between MasterMix for Real-time PCR and Reverse Transcription PCR?
may i follow M.Tevfik Dorak's guide? As i know, he wrote recomendations for Real-Time RT-PCR.
Eh, sorry if my questions are quite stupid

eeee...i mean differense in common reagents =) not Reverse Transcription components

[moleculardude]

Hey Leowe,

I know there are differences between real-time PCR and RT-PCR mastermixes.

I would buy a kit for real-time PCR and RT-PCR and follow the guidlines of the kits. This is the easiest.

If you are doing everything from scratch yourself, I would be more careful. A look at some published research papers in your field should yield some good protocols for these techniques.

Please post back if you have any troubles.

welcome my friend to the foruM!

[Silverwolf]

I bought a ready made master mix from applied biosystems for taqman (its the easiest way, since you are doing taqman)...
Also for RT-PCR ( i do 2 step real time RT PCR) it is easier to use a kit since you need pcr buffer, MgCl2, reverse transcriptase, RNAse inhibitor and primers :S
Its quite many things to find out...