Hi there,
Skipping all the suffering.. I now have my libraries for ChIPs-Seq: good looking peaks, but also a high molecular weight something on the right of the upper marker Worth to notice, that only IPd samples have this peak, inputs do not have it.
PCR conditions:
- inputs ~2-2.5 ng, 12 cycles;
- IPs 0.5-1 ng, 15 cycles - all withing the recommended range in the kit.
Beads carryover? I have noticed that some beads are not sticking to the magnet. But I leave 2.5 uL behind not to aspirate these beads. Repeated treatment on the magnet did not help. I was thinking, may be my beads decomposed and there are tiny speckles I can not separate? I spinned some libraries 15 min 15000 rpm - does not help.
Second size-selection on some libraries diluted to ~100 pg/uL did not help either (may be DNA is too diluted for Ampure beads?)
I found this thread to be very useful,
but as it is quite old, may somebody else can tell me more on what that tail is and how "dangerous" it is for the sequencing? The sequencing facility proposes to do a trail with MiSeq and then if the libraries perform OK, the run on NovaSeq. Reasonable, but superexpensive. May be not worth it (more expensive than the new prep).
ssing did an interesting test. They denatured the samples and then checked them on gel - no HMW tail anymore! Hope to see that with my samples.. But still not getting what it is and how this tail can affect the sequencing.
Thank you all!
Skipping all the suffering.. I now have my libraries for ChIPs-Seq: good looking peaks, but also a high molecular weight something on the right of the upper marker Worth to notice, that only IPd samples have this peak, inputs do not have it.
PCR conditions:
- inputs ~2-2.5 ng, 12 cycles;
- IPs 0.5-1 ng, 15 cycles - all withing the recommended range in the kit.
Beads carryover? I have noticed that some beads are not sticking to the magnet. But I leave 2.5 uL behind not to aspirate these beads. Repeated treatment on the magnet did not help. I was thinking, may be my beads decomposed and there are tiny speckles I can not separate? I spinned some libraries 15 min 15000 rpm - does not help.
Second size-selection on some libraries diluted to ~100 pg/uL did not help either (may be DNA is too diluted for Ampure beads?)
I found this thread to be very useful,
but as it is quite old, may somebody else can tell me more on what that tail is and how "dangerous" it is for the sequencing? The sequencing facility proposes to do a trail with MiSeq and then if the libraries perform OK, the run on NovaSeq. Reasonable, but superexpensive. May be not worth it (more expensive than the new prep).
ssing did an interesting test. They denatured the samples and then checked them on gel - no HMW tail anymore! Hope to see that with my samples.. But still not getting what it is and how this tail can affect the sequencing.
Thank you all!
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