Amplification and Labelling

[Megz24]

Hi!

I am using the Agilent Low input quick amp direct labelling kit and am having very inconsistent amplification and dye incorporation. I am assuming that the problem is occuring with the Transcription mix (contains water,5X trans buffer, 0.1M DTT, NTP Mix, T7 RNA Polymerase and Cy3/Cy5). I've tried to increase the amount of enzyme, dye and incubation time, all of which had no significant effect. I ensure that the enzyme is kept cold until the last possible minute, that the transcription mix is well mixed (inverting several times, gently flicking and spinning down) before adding it to the samples and I keep my samples out of light as much as possible.

I was wondering if anyone else was having consistency issues or may be able to provide any suggestions as to what might be going on with my samples. I am following the protocol exactly as decribed and I'm at a loss for trying to determine what is going on. Any help/suggestions would be greatly appreciated!!

[amjubre]

Hi !

I apologize, I don't speak English very well !

I have sometimes same problem but I don't know where from it can come.
Did you find the solution?
And can you explain your "inconsistent amplification and dye incorporation".
For me, sometimes I have for exemple 30ng/µl without incorporation dye.

[nallias]

Hello Megz24,

Was you able to figure it out ?
How did you measured amplification and dye incorporation ?