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Old 08-25-2017, 05:44 AM   #1
LozWhite
Junior Member
 
Location: Germany

Join Date: Aug 2017
Posts: 1
Question Adapter multimer problem

Hi all,

I am trying to trouble-shoot a problem I am having with my library prep.

I am preparing samples following Meyer & Kircher (2010) for Illumina sequencing with home-made adapters identical to Illumina's.

Using an input of 200ng of sheared DNA, I am getting very low library yields and strange dimer mutlimer's (see attached bioA results).

I have been using SPRI beads to clean up between steps and after PCR, but the smaller peaks are still coming through, as are some larger peaks.

Any ideas what could be causing this? I have double checked my DNA (definitely 200ng going in, and properly sheared) and my SPRI beads (working as expected on DNA ladders). My next best guess was that the blunt-end repair and/or phosphorylation reaction is not working properly? Perhaps degraded ATP or enzymes?

Any help would be really appreciated.
Attached Images
File Type: jpg 170824_2_EGRAM_Sample11.jpg (50.2 KB, 35 views)
File Type: jpg 170824_2_EGRAM_Sample12 (1).jpg (50.2 KB, 19 views)
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