I am testing the kit Power SYBR Green master mix of A & B for my starters and despite varying the temperature anealing and times do not work for me for any gene (even for housekeeping) conditions are: Activation of the enzyme at 95 10 min, 40 cycles of denat at 95º 15 sec+ 30 sec 50 to 59 ºAneal+ Elong 72 60 sec and melting by default as does the computer! what else I can change? it works because my cDNA amplified in Roche! one of my primers sequence is: F-and R-GGACATCTAAGGGCATCACAG GACACGGACAGGATTGACA
Are using the Power SYBR mix on a Roche instrument? Do you see anything in the amplification curves or melt curves? Also, are you doing a gradient for the annealing step? For the Power SYBR mix, we would recommend to redesign the primers slightly so that the Tm’s of both are closer to 60C (right now they are ~ 56 and 54C). Then use a two-step protocol: 95 C for 10 min (for activation of Taq) Then 40 cycles of: 95C for 15 sec 60C for 1 min How much template did you use? Is this a new cDNA prep, something frozen that worked before? Let us know and we'll be able to better answer your question.
***** thank you very much for your reply I use the kit in the Step One Plus but it turned out it was bad production (compare it to the FAST Sybr Green Master Mix A & E) and the latter if it worked!!
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I am testing the kit Power SYBR Green master mix of A & B for my starters and despite varying the temperature anealing and times do not work for me for any gene (even for housekeeping) conditions are: Activation of the enzyme at 95 10 min, 40 cycles of denat at 95º 15 sec+ 30 sec 50 to 59 ºAneal+ Elong 72 60 sec and melting by default as does the computer! what else I can change? it works because my cDNA amplified in Roche! one of my primers sequence is: F-and R-GGACATCTAAGGGCATCACAG GACACGGACAGGATTGACA
Are using the Power SYBR mix on a Roche instrument? Do you see anything in the amplification curves or melt curves?
Also, are you doing a gradient for the annealing step? For the Power SYBR mix, we would recommend to redesign the primers slightly so that the Tm’s of both are closer to 60C (right now they are ~ 56 and 54C). Then use a two-step protocol:
95 C for 10 min (for activation of Taq)
Then 40 cycles of:
95C for 15 sec
60C for 1 min
How much template did you use? Is this a new cDNA prep, something frozen that worked before? Let us know and we'll be able to better answer your question.
***** thank you very much for your reply I use the kit in the Step One Plus but it turned out it was bad production (compare it to the FAST Sybr Green Master Mix A & E) and the latter if it worked!!