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Hi all,
I have a question about the immobilization step in the 454 library prep process. When checking the final single stranded library on an RNA pico chip I see a prominent band around 100 base pairs. The presence of this band seems to correlate with poor/low abundance library generation. I've only seen this band on an RNA pico chip. I've done some troubleshooting and have found that if you wash and elute from naked immobilization beads (mock reaction) you'll also see the same band. This suggests that it's not adapters somehow leaking through, but something inherent to the immobilization beads even after they're washed as indicated. Does anyone know if there's any sort of single stranded fragment pre-bound to these beads that somehow gets competed off (and washed away) in the presence of an abundant library, but remains bound and eluted when there isn't enough of a library around? I'm at a loss as to what this band may be. Thoughts?
thanks
I have a question about the immobilization step in the 454 library prep process. When checking the final single stranded library on an RNA pico chip I see a prominent band around 100 base pairs. The presence of this band seems to correlate with poor/low abundance library generation. I've only seen this band on an RNA pico chip. I've done some troubleshooting and have found that if you wash and elute from naked immobilization beads (mock reaction) you'll also see the same band. This suggests that it's not adapters somehow leaking through, but something inherent to the immobilization beads even after they're washed as indicated. Does anyone know if there's any sort of single stranded fragment pre-bound to these beads that somehow gets competed off (and washed away) in the presence of an abundant library, but remains bound and eluted when there isn't enough of a library around? I'm at a loss as to what this band may be. Thoughts?
thanks