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Old 11-06-2009, 06:42 AM   #1
Cambridge454
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Default emPCR additive

I have not once been able to fully dissolve the precipitate in the emPCR additive, even after heating to 55C. It does not seem to affect the sequencing results but I have noticed especially with the LV reaction that this precipitate seems to reappear during bead recovery and makes the bead pellet very difficult to break up after centrifugation.
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Old 11-06-2009, 09:01 AM   #2
guz
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We have also seen a crystallized precipitate after heating GC additive. I suggest spinning down the tube after heating and only removing GC additive that is found above the pellet. We have found GC additive to aggregate around the bead pellet during LVE breaking, which disrupts washing and enrichment.
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Old 11-06-2009, 02:19 PM   #3
454Sequencing
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Hi Cambridge454,

Allow me to lend a hand. Are you familiar with Technical Bulletin 011-2009? If not, it is available on the my454 customer site
(http://www.454.com/my454) under the "Technical Bulletins" section.

Section 3.1 of that document, Step 1c. reads as follows:

"If a precipitate is observed after thawing the tube of emPCR Additive, vortex the tube well to dissolve it.

If precipitation remains in the emPCR Additive after vortexing, heat the reagent
to 55C in a heat block for up to five minutes to aid in dissolving. If residual
precipitate remains, briefly centrifuge the tube and use the supernatant in the
preparation of the Live Amplification Mix."

In other words, the recommended course is to avoid carrying over residual precipitate, as suggested by guz.

Best regards,
Jason

Technical Product Manager
454 Life Sciences, A Roche Company
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Old 11-08-2009, 11:28 PM   #4
Cambridge454
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Hi Jason,

Yes, I have read and followed the instructions regarding the additive in the Technical Bulletin 011-2009. There appears to be no precipitate in the Live Amp Mix, it only reappears again after bead recovery in the LVE, which makes the bead pellet very difficult to resuspend and wash.
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Old 11-09-2009, 06:11 AM   #5
pmiguel
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Quote:
Originally Posted by Cambridge454 View Post
Hi Jason,

Yes, I have read and followed the instructions regarding the additive in the Technical Bulletin 011-2009. There appears to be no precipitate in the Live Amp Mix, it only reappears again after bead recovery in the LVE, which makes the bead pellet very difficult to resuspend and wash.
I think I have a fix for this issue you mention (from TCB-09011). I believe the issue derives largely from this section of the protocol (section 3.5 "Bead Recovery):

Quote:
4. Add 10 ml of 1X Enhancing Fluid TW and vortex well to resuspend. If vortexing is not
sufficient to resuspend completely, use a glass rod or a spatula to break bead aggregates.
Note: It is important to fully suspend the bead suspension before adding
isopropanol in the next step to ensure complete mixing and to prevent clumping.
The problem is that 1X Enhancing Fluid TW appears to cause the waxy deposit that makes resuspending the beads so difficult. So the longer you spend trying to resuspend the beads, the worse the clumping situation becomes.

If, instead, you add the 1X Enhancing Fluid TW, vortex briefly and immediately add isopropanol, the formation of precipitate is much less. Clumping then does not seem to be an issue.

--
Phillip
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Old 11-09-2009, 06:37 AM   #6
Cambridge454
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Thanks for the advice. I think this might be my problem, as I usually pour off the isopropanol supernatant, add the 1x Enhancing Fluid TW, vortex, vortex, vortex, break clumps with glass rod, vortex, vortex, vortex, then add isopropanol.
Will give your fix a try.
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Old 11-09-2009, 08:44 AM   #7
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We've found that trying to use a spatula to break up the aggregates makes the clumping worse. Vortex well after additing the Enhancing fluid and then go right to the isopropanol as mentioned above. Better to spend more time vortxing after the isopropanol addition that before. I've managed to get rid of all of the clumps this way.
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Old 06-14-2010, 06:52 AM   #8
Old guy
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Anybody know what the emPCR additive is?
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Old 06-14-2010, 12:16 PM   #9
vlee2
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To avoid clumping I use 4 syringes per 1 cup of oil and have bead recovery rate around 80%. When using this technique I don't have problems with oily and waxy beads, and with clogging coulter counter orifice anymore.
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Old 06-15-2010, 04:43 AM   #10
pmiguel
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Quote:
Originally Posted by Old guy View Post
Anybody know what the emPCR additive is?
I have heard it referred to as a polymerase "stabilizer" by one of the "old ones"[1]. That doesn't tell you much -- even if it is correct, however.

--
Phillip

1. By "old one", I mean someone who ran the instrument back when it was in its original incarnation as the GS-20.
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