Hi everybody,
Could anybody please reflect on what I see on the tape-station report and how it might affect the sequencing?
I have prepared libraries for ChIP-Seq (input, 3 IPs and IgG ctrl). All these libraries were diluted to ~100 - 300 pg/ul to test on the tape-station (HS D1000). All further manipulations and tests were carried out on those diluted libraries. In all except input libraries I see a high molecular weight tail on the right of the upper marker (see attached image). After googling I think the tail looks like the beads carry-over. However, keeping the library prep on the magnet did not help. Neither helped spinning the sample 15 min 15000 rpm. The repeated size-re-selection (0.7X/1.8X) with Ampure XP beads also did not help. Can it be that these libraries are too diluted to re-size select them?
What this HMW tail could be? I do not think I have a problem with overamplification because the initial input DNA concentration was higher (~2.5 ng) than that for IPs and IgG (0.5-1 ng). Though the cycles number was high: 12 for input and 15 for IPs and IgG. The number of cycles is withing the recommendations for the initial DNA concentration. The library was size-selected after PCR.
Any ideas? Will this tail affect the sequencing? What is the origin of this tail?
Thank you :)
Similar discussion is here, quite old though. http://seqanswers.com/forums/showthread.php?t=15224
Still not getting what it is
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