Dear all,
I've been fighting for a while with an ineffective library preparation for Illumina sequencing. Just to introduce the problem, in this protocol I have to pass trough steps of digestion, ligation (NEB T4), purification and amplification on freshly extracted plant DNA.
As you probably imagined by now, I'm not obtaining amplification whatsoever. I've proceeded to all sort of troubleshooting, but no results.
Yet, I'm going to try to make my question as specific as possible:
In order to clean the samples (contamination by SIGMA kit), I precipitated my DNA in EtOH using glycogen prior to library preparation: is anybody aware of problems that glycogen can cause with enzimatic reactions? (digestion/ligation) My doubt right now is that, even if stated otherwise, it could interfere somehow.
If yes, does anybody know how to remove it?
Thanks for any help!
Matteo
I've been fighting for a while with an ineffective library preparation for Illumina sequencing. Just to introduce the problem, in this protocol I have to pass trough steps of digestion, ligation (NEB T4), purification and amplification on freshly extracted plant DNA.
As you probably imagined by now, I'm not obtaining amplification whatsoever. I've proceeded to all sort of troubleshooting, but no results.
Yet, I'm going to try to make my question as specific as possible:
In order to clean the samples (contamination by SIGMA kit), I precipitated my DNA in EtOH using glycogen prior to library preparation: is anybody aware of problems that glycogen can cause with enzimatic reactions? (digestion/ligation) My doubt right now is that, even if stated otherwise, it could interfere somehow.
If yes, does anybody know how to remove it?
Thanks for any help!
Matteo
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