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Our beads prepared with our samples stick fine. Only those made with Control DNA did not stick. When we tried to figure out what the FAS was doing differently so that everything worked that he tried, even the reagents we thought were bad, we finally realized it was different DNA. Specifically AB's control. We have no problems with beads sticking to slides now.
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Do we want to posit one or more mechanisms to explain this? Here are some
(1) The control DNA was degraded or otherwise defective -- nothing to amplify, nothing to tail. The result pre-EZBead would have been: no beads after enrichment. But EZBead has a fairly high "void" bead number -- hundreds of millions for the E80. Were enrichment yields low with the control DNA, DNADEB?
(2) The control DNA has a detergent in it that interferes with binding and somehow persists through ePCR and enrichment.
Other possibilities?
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PhillipComment
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This is APPLIED BIOSYSTEM #A12126 dh10B SOLiD Library Standard that we were told to use when testing the EZ bead system.
It works for qPCR and was not old.
A titration of 3 different amounts is suggested and the enrichment yields were as expected.
I would assume that what they sold us was not defective and had no inhibiting materials.
DebComment
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The bottom line is that our samples we prepare work well and stick to the slide. We have not wasted any more time on why their control does not work.Comment
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Thanks for posting this follow-up. I admire your focus. Sadly I do not share it...
I think terminal transferase requires a blunt double-stranded end to function. Maybe the oligo hybed to the beads resulting from the control DNA did not produce a blunt end, or did not anneal well.
Did the control DNA beads clump at all during tailing?
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PhillipComment
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would have to ask the tech about the clumping. Then they should not have suggested to use it if it wasn't going to work.Comment
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