Hello all,
I'm making fragment librairies on a Solid 5500XL, using LifeTech protocols and standard kits) and i am experiencing troubles in my last lib preps: using mouse DNA, i have no ligation of adaptators and barcodes or incomplete ones.
Strangely, one sample worked perfectly when other 6 didn't ligate at all; one sample showed incomplete ligation with two peaks easily distinguished on the Bioanalyzer.
I wonder if a product in the elution solution used at the DNA extraction step could perturb one of the previous step before it. Preferentially the end-repair because if an extraction reagent remains in my DNA eluate, i'll get rid of it while performing the size selection, and the only step chemical step done before that is the end-repair one. By the way, the shearing goes perfectly well.
A last information is that the only sample that had no issue throughout the whole process was the non-treated/non-irradiated control animal when the other had UV irradiations and chemical treatments.
Thank you for your help,
Durandg.
I'm making fragment librairies on a Solid 5500XL, using LifeTech protocols and standard kits) and i am experiencing troubles in my last lib preps: using mouse DNA, i have no ligation of adaptators and barcodes or incomplete ones.
Strangely, one sample worked perfectly when other 6 didn't ligate at all; one sample showed incomplete ligation with two peaks easily distinguished on the Bioanalyzer.
I wonder if a product in the elution solution used at the DNA extraction step could perturb one of the previous step before it. Preferentially the end-repair because if an extraction reagent remains in my DNA eluate, i'll get rid of it while performing the size selection, and the only step chemical step done before that is the end-repair one. By the way, the shearing goes perfectly well.
A last information is that the only sample that had no issue throughout the whole process was the non-treated/non-irradiated control animal when the other had UV irradiations and chemical treatments.
Thank you for your help,
Durandg.