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Old 03-03-2011, 12:11 PM   #1
pmiguel
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Default Beads not sticking to slides

A while back, DNADEB posted in this thread:

http://seqanswers.com/forums/showthread.php?t=8057

to say that beads tailed with new deposition kits were not sticking to slides. We recently have had this problem as well. Anyone else?

By not sticking, I mean in a catastrophic way--the beads just completely pulled away from the slide before it could even be mounted on the instrument.

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Old 03-09-2011, 07:52 AM   #2
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We worked with FAS and it started working again. Even working one on one at the same time, the FAS and tech could never spot any differences! Many Many controls were run including tubes, slides, reagents, water etc. Talked to another lab with this problem as well --haven't heard back.
So we are in business again!
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Old 03-11-2011, 05:33 AM   #3
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Okay thanks for your reply.
Our FAS replaced our deposition kit and now everything seems back to normal for us as well.

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Old 03-11-2011, 06:14 AM   #4
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This is something to do with the 3 prime modification. The enzyme might be faulty. Before you even get to the bead deposition stage you should look at the beads and they should clump together. After doing the terminal transferase reaction check for clumpiness. You can't miss it. If the beads don't appear clumpy you have a problem.

This should save you wasting time and beads.
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Old 03-11-2011, 06:54 AM   #5
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Probably, yes. Although sometimes we have problems with beads being too clumpy--as that can interfere with deposition also.

BTW, any idea what the basis of the binding of the beads to the slide is? During training in Foster City the trainers went all inscrutable when asked. So I presume they are using someones IP with promise of keeping it secret. Irritating though.

Terminal transferase just takes a double stranded end and adds a bunch of bases to it--from dNTPs, I presume. Why would that cause beads to "clump" anyway? Physical tangling, or something else?

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Old 03-11-2011, 07:03 AM   #6
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So with the beads being too clumpy. I have used the new protocol of splitting the E80 beads in two, 3 lots of declump 1, combine and 3 lots of declump 3.

We have worked out that the best deposition, ie best quality QV is off 240K per panel. We have got a few slides with 270K per panel but the QV values are low.

I setting up some experiments of the same library, beads but with a 240K depo and a 170K depo. We will then know the difference. We suspect the lower density give higher QV. Sorry we know this but we need to show the experiment with no other variables to get the point across.

Strangely quads and octs don't have any depo problem. This is due to the lower bead density.

AB don't acknowledge this but I don't care. We continue on regardless.
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Old 03-11-2011, 07:21 AM   #7
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Very interesting.

We basically gave up trying to get above ~210K/panel--not because the QVs were lower, but because nothing we did seemed to get us there. For our current run we deposited 1 billion beads and the result is just under 500 million usable beads total (208K/panel). We tried depositing more, but then we were seeing lots of slides breaking--no idea why, but it doesn't take too many occurrences of losing a run 10 days in to dim the appetite for experimentation.

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Old 03-29-2011, 08:02 AM   #8
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Quote:
Originally Posted by niceday View Post
Strangely quads and octs don't have any depo problem. This is due to the lower bead density.

AB don't acknowledge this but I don't care. We continue on regardless.
This phenomenon has been acknowledged (i.e. the higher bead:volume ratio for E80 causes a greater degree of clumping)! - this is why the modified protocol for E80 scale was written!
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Old 03-29-2011, 08:05 AM   #9
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Officially?
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Old 03-29-2011, 08:12 AM   #10
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Did they give you a modified protocol?
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Old 03-29-2011, 08:15 AM   #11
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not directly. We now have one and it works better. Still not close to 300K per panel. We know what the problem is. An official explanation would be useful and some indiciation that the problem will have been solved before the 5500 hit the shelves.
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Old 03-29-2011, 08:26 AM   #12
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That's odd - our FAS gave us an official recommendation a few months ago...
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Old 03-30-2011, 10:47 AM   #13
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Is your FAS named Merlin, perchance? I mean, if he is living backwards through time that could explain how he had access to a protocol not released to the rest of us.

Seriously, though, we get modified protocols of one sort or another all the time. Hard to know exactly what is being discussed here. What differences were there in the new protocol?

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Old 04-08-2011, 07:20 AM   #14
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It turns out that the REAL reason ours failed has to do with the CONTROL! When the FAS came to work at our site with our tech and new reagents etc, everything worked. These were our samples. However, later we came to find out it is actually the CONTROL DNA that they supply that does not work. The tech used it when she was starting to use the EZ Bead system and the manual suggested starting with their Control DNA.
WELL.....................
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Old 04-08-2011, 08:17 AM   #15
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The Control DNA would still not explain beads coming off the slide.
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Old 04-08-2011, 09:13 AM   #16
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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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Old 04-08-2011, 10:07 AM   #17
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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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Old 04-08-2011, 10:57 AM   #18
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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.
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Old 04-08-2011, 10:58 AM   #19
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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.
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Old 04-08-2011, 11:33 AM   #20
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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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