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Old 10-17-2014, 03:54 AM   #1
jdp
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Default Ampure XP clumping

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
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Old 10-17-2014, 03:19 PM   #2
kerplunk412
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I have only ever experienced clumping when drying extensively, and even then the clumps can be broken up with vigorous pipetting.

Don't take offense to this question, but you aren't trying to resuspend with the tubes still on the magnetic rack, right? Another thought is that you may have too much DNA and that is causing clumping, but I think that is unlikely.

It may help to add a small amount of Tween to your elution buffer, something like 0.05-0.1%.

If you haven't already, I would recommend contacting Beckman tech support. I had a very good experience with them.

Last edited by kerplunk412; 10-17-2014 at 03:24 PM. Reason: More thoughts
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Old 10-20-2014, 06:22 AM   #3
jdp
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@kerplunk412

Thanks for your reply!
That extensive drying could lead to clumping is clear and that's why I reduced the drying time to only about 4 minutes. Thus I am more concerned about possible EtOH carryover and downstream PCR inhibition.

Sure, I remove the plate from the stand! But I also wondered whether the effect might depend on the kind of magnetic stand one uses. I have several different designs available and I thought it might be that the stand I use is too strong so the beads clump from this. I will have to try this out.

Beckman tech also thought the amount of DNA might be too high. I see that this could lead to clumping but I certainly don't have to much DNA in it.

I will look into the Tween idea. You add this to the elution buffer by default?

As it seems this problem is not really widespread.
I will thus contact Beckman tech again.

jdp
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Old 10-20-2014, 08:02 PM   #4
austinso
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If you have a lot of protein, like BSA, you will get clumping.

I'm also wondering how much CTAB is carried-over...it is a hydrophobic cationic detergent, so it will bind to the carboxylated bead surface. Also, I don't think the CTAB prep is necessarily "clean" with just a chloroform extraction, so a lot of protein carryover is possible...I thought an SDS post-clean is usually required...?

FWIW...
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Old 10-22-2014, 05:09 AM   #5
jdp
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@austinso
thanks for your reply. You are right, the extracted DNA is certainly not entirely clean. I have no SDS step in my protocol but use B-mercapt-EtOH and PVP.

I notice however that the clumping problem occurs in the negative pcr controls as well. Thus any proteins or impurities from the extract can not be (the only) cause.

As I said, I use BSA in the PCR. Did you experience this effect on BSA yourself or where is this information from?

Thanks!
jdp
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Old 10-22-2014, 08:49 AM   #6
austinso
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Quote:
Originally Posted by jdp View Post
As I said, I use BSA in the PCR. Did you experience this effect on BSA yourself or where is this information from?
Personal experience. Restriction digests with BSA, among others.

A.
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Old 10-22-2014, 07:25 PM   #7
han526
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Default avoid completely drying beads

BSA and other impurities can be the problem. Even pure beads can have problem when completely dried. The best is just only let ethanol evaporate enough but still keep the beads moisturized. Avoid beads cracking. The residual ethanol doesn't really affect down stream application, unless you run a submerged gel.

BTW, you can use PCRClean from Aline Biosciences to direclty replace Ampure.

Quote:
Originally Posted by jdp View Post
@austinso
thanks for your reply. You are right, the extracted DNA is certainly not entirely clean. I have no SDS step in my protocol but use B-mercapt-EtOH and PVP.

I notice however that the clumping problem occurs in the negative pcr controls as well. Thus any proteins or impurities from the extract can not be (the only) cause.

As I said, I use BSA in the PCR. Did you experience this effect on BSA yourself or where is this information from?

Thanks!
jdp
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Old 10-23-2014, 04:07 PM   #8
kerplunk412
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Quote:
Originally Posted by jdp View Post
@kerplunk412


I will look into the Tween idea. You add this to the elution buffer by default?


Nope, it was just an idea to help prevent the clumping. I have never had a problem with water or resuspension buffer (10 mM TRIS). The BSA sounds like the most promising lead now. I assume you will try a reaction without BSA, please let us know how it turns out!
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Old 10-27-2014, 03:35 AM   #9
jdp
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A quick test with a few reactions without BSA was very promising! No clumping at all. So it seems this was indeed the reason for the clumping. In case I observe the clumping again without BSA during library prep I will post it here!

Thanks for your help!
jdp
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Old 01-20-2015, 01:56 AM   #10
JMA
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We have encountered the same issue with BSA and AMPure beads that results in clumping. Unfortunately we need a certain amount of BSA for a reproducible fragmentation with our fragmenation enzyme mix.

Does anyone know about a way to reduce the effect of BSA clumping with the beads. I found that maybe adding Tween20 or SDS might help. Does anyone have experience on this?
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Old 07-27-2015, 04:22 AM   #11
Vasco
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Hello every one,

I did run into the same issues. Im working on COi amplicon sequencing with the Illumina sequencers.

I tried the same protocols as usually, but added BSA in the PCR step. After PCR we did a size selection with SPRIselect (more or less the same as Ampure XP), and experienced the described resuspension problems. Also with pipeting, short vortexing and incubation for 1 hour in a fridge (4C) the beats would not resuspended.

The Backman support claimed its due to pellet drying after EtOH wash or to high PCR product concentrations, wich is not the case (it does work just fine with PCRs without BSA).

The size selection did work more or less (some primer clouds remained in the gel picture, and the PCR product was also present). So we pooled the 10 size selected PCR replicates into one library, and did the size selection with SPRIselect again. This time the pellets resuspended just fine, but the beats did adhere not well to the magnets in the step before (when removing the SPRIselect buffer). They formed lumps and got into solution when pipetting. However, the size selection did still work (concentrations look good, but we have not run the gel jet).

I am not really sure how much BSA is still in the final library, as the beats still behaved untypical. I probably will run the library trough a Reaction clean up kit, just to make sure no BSA remains in the solution.

Best regards
Vasco
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Old 05-30-2017, 05:50 AM   #12
JBKri
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Yesterday I experienced a similar problem, but the clumping happened in the first step, at the mixing of the bead solution and the DNA. This was HMW genomic DNA (from a phenol-chloroform extraction) which we do not want sheared, so I did not attempt to vigorously mix. I had to proceed with the ethanol washes with some lumps that could not be homogenized. In the final elution I was able to dissolve the lumps after a long incubation, and in the end I seem to have recovered a decent amount of HMW DNA. BUT, the Nanodrop ratios are worse than before the cleanup! Perhaps the lumps prevented efficient ethanol washes?

Jon
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Old 06-05-2017, 07:30 AM   #13
anjama
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Quote:
Originally Posted by JBKri View Post
Yesterday I experienced a similar problem, but the clumping happened in the first step, at the mixing of the bead solution and the DNA. This was HMW genomic DNA (from a phenol-chloroform extraction) which we do not want sheared, so I did not attempt to vigorously mix. I had to proceed with the ethanol washes with some lumps that could not be homogenized. In the final elution I was able to dissolve the lumps after a long incubation, and in the end I seem to have recovered a decent amount of HMW DNA. BUT, the Nanodrop ratios are worse than before the cleanup! Perhaps the lumps prevented efficient ethanol washes?

Jon
I actually just came across this thread trying to figure what is clumping my beads, but in my case I was using them to extract dna from digested tissue in lysis buffer. Based on your comment and the comments above, my clumping could be the result of HMW dna and residual proteins. The clumps finally break apart in the water elution, and I was still able to get good concentrations with good nanodrop readings. If you're doing yours on a magnetic stand, consider doing 3 ethanol washes. I've been doing 3 without any problems to help minimize salt carryover from the bead solution, which would cause low 260/230 ratios. I'm not 100% sure, but I believe ethanol also absorbs more at 230 than at 260, in which case ethanol carryover would also cause low 260/230 ratios. I pipette every last drop of ethanol out and let my beads dry until they crack to make sure absolutely no ethanol remains.
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