Hi to all!
I'm new at aCGH Array and I need some advice.
I'm working following Agilent Oligonucleotide Array-Based CGHfor Genomic DNA Analysis using the Enzimatic Labeling protocol , from Agilent.
I'm starting from 500 ng of good quality gDNA and I perform a digestion and a labeling step as it is described on the protocol. The issue is that, when I mesure the degree of labeling and specific activity of the samples (labeled with Cy5) and the references (labeled with Cy3) I'm not sure if I'm getting good results...
The results I get are the following ones :
[B]Samples [ ] / Yield ug / pmol/ul /DOL (%) /sp.act
S1 (Cy5) = 196 ng/ul, 4.5ug, 5.9, 1.02%, 30.1
S2( Cy5)= 217,9 ng/ul, 5ug, 6.8, 1.06%, 31.2
R1 (Cy3)= 211,6 ng/ul, 4.4ug, 8.6, 1.38%, 40.6
R2 (Cy3)= 175,6 ng/ul, 3.68ug, 7.5, 1.45%, 42.71
And, on the protocol, it says that the values should be :
Tabla 27. Expected Yield and specific activity after labeling and clean-up (pagina 55 manual)
Input gDNa (ug) Yield (ug) sp.act Cy3 (pmol/ul) sp.act Cy5 (pmol/ul)
0,2-0,5/ 2,5-3/ 15-25/ 15-20
0,5/ 5-7/ 25-40/ 20-35
3/ 7-10/ 35-55/ 25-40
The concentrations ara Ok and I think that the pmol/ul are also ok, but the yield stil being a bit low and the specific activity of Cy3 seems to be low....any advice???
I would like to see some examples of values that have been used to hybridize and that have given good results. I don't know if i can hybridize these samples or not...
Thanks a lot in advance