Hi All,
I'm having a weird 350 bp band showing up in my ChIP-seq Input libraries. I am using the Illumina protocol. After size selecting 175-225 bp and then PCRing, a 350 bp band has shown up on my gel in addition to my 200bp band. Does anyone know where this is coming from?
My theory is that after the ligation of the adapters when I clean up the reaction, I elute from the min-elute column with 55 degree Elution Buffer. This higher temp may been generating a 350 bp single stranded DNA that is migrating along with my 175-225 dsDNA. Does this seem like a plausible explanation? I have also been digesting the gel fragments at room temp to prevent GC bias like has been suggested here on the forum.
Thanks for the help and I am posting pics of the pre and post pcr gels.
Mark
I'm having a weird 350 bp band showing up in my ChIP-seq Input libraries. I am using the Illumina protocol. After size selecting 175-225 bp and then PCRing, a 350 bp band has shown up on my gel in addition to my 200bp band. Does anyone know where this is coming from?
My theory is that after the ligation of the adapters when I clean up the reaction, I elute from the min-elute column with 55 degree Elution Buffer. This higher temp may been generating a 350 bp single stranded DNA that is migrating along with my 175-225 dsDNA. Does this seem like a plausible explanation? I have also been digesting the gel fragments at room temp to prevent GC bias like has been suggested here on the forum.
Thanks for the help and I am posting pics of the pre and post pcr gels.
Mark
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