Hi everybody!
I'm in the course of library prep for 16S community analysis on Illumina MiSeq. The DNA originates from the microbial community of small water flea.
Extraction is a classic CTAB, Chlorophorm method. No column.
Yield is sufficient and pretty pure.
For the cleanup after PCR (first PCR and after Indexing) I use Ampure XP beads. Volume: 0.8x, wash twice with 190ul EtOH 80%. My problem is that after drying from EtOH, when I try to elute the DNA with Tris, the beads clump and some stick to the wall of the microtube and to the pipette tip etc... Sometimes it worked easily and sometimes it just sticks and I can not bring it back into suspension. I tried everything... I had the impression, that it depends on the drying time so I tried to go down to 4 minutes at RT but the problem is still there. I use only lo bind plastic.
It is so annoying because I can not understand where the Problem lies and what to try...
Did anybody experience similar behaviour or have an Idea what could be the reason? I am afraid to shorten the drying even more due to the risk of EtOH carryover...
Thanks a lot!
jdp
I'm in the course of library prep for 16S community analysis on Illumina MiSeq. The DNA originates from the microbial community of small water flea.
Extraction is a classic CTAB, Chlorophorm method. No column.
Yield is sufficient and pretty pure.
For the cleanup after PCR (first PCR and after Indexing) I use Ampure XP beads. Volume: 0.8x, wash twice with 190ul EtOH 80%. My problem is that after drying from EtOH, when I try to elute the DNA with Tris, the beads clump and some stick to the wall of the microtube and to the pipette tip etc... Sometimes it worked easily and sometimes it just sticks and I can not bring it back into suspension. I tried everything... I had the impression, that it depends on the drying time so I tried to go down to 4 minutes at RT but the problem is still there. I use only lo bind plastic.
It is so annoying because I can not understand where the Problem lies and what to try...
Did anybody experience similar behaviour or have an Idea what could be the reason? I am afraid to shorten the drying even more due to the risk of EtOH carryover...
Thanks a lot!
jdp
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