And by "OLD reagent" you mean only Terminal Transferase, or the entire deposition kit? (This is v4 "XD" reagents, right?)
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Phillip
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Phillip
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No I mean the kit we purchase to use for 3 PLUS and the IKA. It is packaged differently and the protocol is different. It is likely the same but the old dep kit is what works.Comment
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So, just to be clear, you tried 3 times, with three kits, to deposit EZBeads. But the beads would not stick to the slide. (Were the kits that failed the XD kits?) Then you found that by using an old 3+ deposition kit, it did work--even with the EZBeads?
If so, that sounds like a 3+ vs. XD deposition kit issue.
I have heard that XD deposition is very sensitive to detergents (whereas, apparently the v3+ deposition kits were not.) Could that be the issue?
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PhillipComment
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yes we have visited the detergent issue!
OLD Beads from the freezer stick fine when put in two quads of the same slide. Beads modified with the new kit and placed in the other two quads do not. UNLESS they are then RE-treated with the term transf from old kit.Comment
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Are all the new deposition kits "XD" kits?
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PhillipComment
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dont know yet. We have a huge email ready to go out soon...
lets see what they sayComment
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I meant are all the kits you describe as "new", that have failed, are they all XD kits?Comment
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Yes they areComment
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Try to declump before adding Terminal Transferase. It seems helpful.Comment
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Do you mean helpful in getting beads to stick, or helpful for preventing bead clumping?Comment
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I will put my two cents in here as well. EZ Bead works quite nicely as Vanessa pointed out. I don't have any experience with the IKA, so I can't compare the two directly. I have made different library preps for all three scales and there are only two problems that jump out at me.
1: the @#!% Enricher doors are not designed well. Sometimes they can lose contact with the port and will effectively end a run immediately with the only way to proceed being to go back and analyze the log file, find the last step and then to have the materials on hand to proceed via manual enrichment. This will be fixed though within the next two weeks if I understood my FAS correctly...
2: the programs don't give the correct time to completion. Not a big deal, but it would be nice to have it right.
For the tube problems that some people have had, I do all oil prep for the emulsions in 50ml conical tubes from VWR. The oil was still good 10 days later based on a subsequent library prep.
And last, but not least, the only problem I have had with terminal transferase was that...there is not enough. A normal E80 will almost wipe one out. One thing that I have noticed, maybe I am imagining things, is that the speed at which I run the rotator does play a part in how well term. transferase works. Briefly, it doesn't like too fast.
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Ok, now I'm going to be paranoid about the Enricher doors, as well as the syringe. Not that I've had issues with the doors but now I'll worry just for good measure.
The incorrect times are a pet peeve of mine as well, but since our stuff works so well it practically glows, I'll not complain about it.
The whole 3'mod thing not working is also worrisome. What are the lot numbers that AB is saying are not good?
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Interesting about the terminal transferase reaction rotator. We use the (ironically?) named "Lab Quake" for most rotations. Nothing warm blooded would think of it as going "too fast".
I wonder if Invitrogen just had a poorly mixed batch of Terminal Transferase? Ours might have been too concentrated, DNADEB's not concentrated enough and VanessaS's just right. (Complete speculation on my part, of course. I don't know that the Terminal Transferase is even sourced from Invitrogen...)
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PhillipComment
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Woohoo, Goldilocks and the three Bears, NGS style!Comment
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i doubt it's the TT.
My guess would be the bead linker. This used to degrade in the manual ePCR days from numerous freeze-thaws. It's much more likely a culprit than the TT.
I asked 3 other sites and the FAS, and there is no word of bad lots yet.Comment
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