Problem with AMPure purification

JanaSEQ · 07-25-2014, 05:09 AM

Hi,
Maybe someone has faced the same problem, as I am:

I am using Agencourt AMPure XP to clean my PCR-amplicons from PCR-reaction mixture. I am using AMPure at 1x sample volume, to get rid of all the primers.
In the last few months I started noticing, that the AMPure magnetic beads do not pellet properly on the tube wall after the addition of Elution Buffer (10 mM Tris-HCl, pH 8,5). The DNA concentrations of amplicons also decreased. I tried to elute my amplicons in MQ water and it worked well, I also obtained a bit higher DNA concentrations. But I am still wondering, what is wrong with the elution using EB. I checked my Ethanol and tried to use 80% instead of 70%, it did not change anything. I also tried to use different lab plastic, magnet plate and ordered fresh EB and AMPure. Nothing worked and I still got this „haze“ instead of properly pelleted beads.

Maybe someone could help me, what am I doing wrong?

rnaeye · 07-25-2014, 07:47 AM

Are you using the same PCR polymerase/buffer. I know that having proteins like BSA in the sample causes haze during Ampure cleanup. I cannot explain why your problem disappears with MQ water though.

JanaSEQ · 07-28-2014, 12:19 AM

I am using Maxima HotStart PCR Master Mix. I tried to leave out ethanol purification steps (just to see, what happenes) and it worked - the haze was gone. But I can´t do that with my real samples..

bilyl · 07-28-2014, 07:01 PM

Quote:

Originally Posted by JanaSEQ

Hi,
Maybe someone has faced the same problem, as I am:

I am using Agencourt AMPure XP to clean my PCR-amplicons from PCR-reaction mixture. I am using AMPure at 1x sample volume, to get rid of all the primers.
In the last few months I started noticing, that the AMPure magnetic beads do not pellet properly on the tube wall after the addition of Elution Buffer (10 mM Tris-HCl, pH 8,5). The DNA concentrations of amplicons also decreased. I tried to elute my amplicons in MQ water and it worked well, I also obtained a bit higher DNA concentrations. But I am still wondering, what is wrong with the elution using EB. I checked my Ethanol and tried to use 80% instead of 70%, it did not change anything. I also tried to use different lab plastic, magnet plate and ordered fresh EB and AMPure. Nothing worked and I still got this „haze“ instead of properly pelleted beads.

Maybe someone could help me, what am I doing wrong?

The "haze" sometimes occurs if you incubate the bead mixture for too long. I've seen it a lot but only when I do "in-bead" enzymatic reactions and cleanup afterward with SPRI buffer (PEG/NaCl). I've also seen it if I overdry my beads. Maybe something to think about.

JanaSEQ · 07-28-2014, 11:48 PM

Thanks bilyl!
I have tried different incubation times. I have also tried to elute amplicons right after I removed ethanol and also tried to let the beads dry completely, before I added the elution buffer. I did not notice any changes.

silviap · 08-03-2014, 10:35 AM

I' m using AMPure on a Beckman Platform for many years and I used water MQ as elution buffer without any problem. Are you using the original magnet plate?

JanaSEQ · 08-10-2014, 11:52 PM

I am using DynaMag magnet plate (Life Technologies). I am not using MQ water, because sequencing company where we send our samples prefers PCR amplicons in Tris-HCl buffer.

dtm2451 · 08-11-2014, 08:48 AM

I'm not certain what might be going on here, but if MQ seems to work for the elution, could you just elute in water and then dilute in elution buffer?

JanaSEQ · 08-12-2014, 12:09 AM

Thank you Dan (dtm2451)!

I will try this out.

But I still would like to know, what could have happned with the purification. My last idea is, that maybe it´s the hot weather, that causes such abnormalities (air conditioner in my lab does not work properly and it is up to 30C/86F) But i refrigirate Ampure (and then bring it to room temperature immediately before using) I also tried to refrigerate ethanol and elution buffer, unfortunately it did not help.

JanaSEQ · 09-02-2014, 04:09 AM

My problem seems to be gone now! During last few Ampure purifications I did not notice haze any more. I did not change anything and so I think the haze was a result of way too hot working conditions. Fortunately is summer over now!

dtm2451 · 09-02-2014, 07:41 AM

My lab also gets fairly hot in the Summer due to inefficient AC. Perhaps that was the issue as I'm no longer having SPRI trouble either. I don't like the heat explanation... I really don't get why that would inhibit bead binding, but it might be true. Either way, I'm glad you are no longer having issues either!

docphil · 09-22-2014, 04:09 PM

My Lab uses AMPure beads at 0.7x to remove primer-dimer and any junk below our target after barcoding and final pooling for MiSEQ. We elute into TE and then run the eluted DNA through a QiaQuick spin column to remove any residual beads. You can also adjust the amount of EB you spin through the column if you need a higher DNA concentration. Might be a bit redundant but hopefully it helps!

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07-25-2014, 05:09 AM	#1
JanaSEQ Junior Member Location: Estonia Join Date: Oct 2013 Posts: 8	Problem with AMPure purification Hi, Maybe someone has faced the same problem, as I am: I am using Agencourt AMPure XP to clean my PCR-amplicons from PCR-reaction mixture. I am using AMPure at 1x sample volume, to get rid of all the primers. In the last few months I started noticing, that the AMPure magnetic beads do not pellet properly on the tube wall after the addition of Elution Buffer (10 mM Tris-HCl, pH 8,5). The DNA concentrations of amplicons also decreased. I tried to elute my amplicons in MQ water and it worked well, I also obtained a bit higher DNA concentrations. But I am still wondering, what is wrong with the elution using EB. I checked my Ethanol and tried to use 80% instead of 70%, it did not change anything. I also tried to use different lab plastic, magnet plate and ordered fresh EB and AMPure. Nothing worked and I still got this „haze“ instead of properly pelleted beads. Maybe someone could help me, what am I doing wrong?

07-25-2014, 07:47 AM	#2
rnaeye Member Location: East Cost Join Date: May 2011 Posts: 79	Are you using the same PCR polymerase/buffer. I know that having proteins like BSA in the sample causes haze during Ampure cleanup. I cannot explain why your problem disappears with MQ water though.

07-28-2014, 12:19 AM	#3
JanaSEQ Junior Member Location: Estonia Join Date: Oct 2013 Posts: 8	I am using Maxima HotStart PCR Master Mix. I tried to leave out ethanol purification steps (just to see, what happenes) and it worked - the haze was gone. But I can´t do that with my real samples..

07-28-2014, 11:48 PM	#5
JanaSEQ Junior Member Location: Estonia Join Date: Oct 2013 Posts: 8	Thanks bilyl! I have tried different incubation times. I have also tried to elute amplicons right after I removed ethanol and also tried to let the beads dry completely, before I added the elution buffer. I did not notice any changes.

08-03-2014, 10:35 AM	#6
silviap Junior Member Location: Milan - Italy Join Date: Aug 2014 Posts: 3	I' m using AMPure on a Beckman Platform for many years and I used water MQ as elution buffer without any problem. Are you using the original magnet plate?

08-10-2014, 11:52 PM	#7
JanaSEQ Junior Member Location: Estonia Join Date: Oct 2013 Posts: 8	I am using DynaMag magnet plate (Life Technologies). I am not using MQ water, because sequencing company where we send our samples prefers PCR amplicons in Tris-HCl buffer.

08-11-2014, 08:48 AM	#8
dtm2451 Junior Member Location: San Francisco, Ca Join Date: Mar 2013 Posts: 9	I'm not certain what might be going on here, but if MQ seems to work for the elution, could you just elute in water and then dilute in elution buffer?

08-12-2014, 12:09 AM	#9
JanaSEQ Junior Member Location: Estonia Join Date: Oct 2013 Posts: 8	Thank you Dan (dtm2451)! I will try this out. But I still would like to know, what could have happned with the purification. My last idea is, that maybe it´s the hot weather, that causes such abnormalities (air conditioner in my lab does not work properly and it is up to 30C/86F) But i refrigirate Ampure (and then bring it to room temperature immediately before using) I also tried to refrigerate ethanol and elution buffer, unfortunately it did not help.

09-02-2014, 04:09 AM	#10
JanaSEQ Junior Member Location: Estonia Join Date: Oct 2013 Posts: 8	My problem seems to be gone now! During last few Ampure purifications I did not notice haze any more. I did not change anything and so I think the haze was a result of way too hot working conditions. Fortunately is summer over now!

09-02-2014, 07:41 AM	#11
dtm2451 Junior Member Location: San Francisco, Ca Join Date: Mar 2013 Posts: 9	My lab also gets fairly hot in the Summer due to inefficient AC. Perhaps that was the issue as I'm no longer having SPRI trouble either. I don't like the heat explanation... I really don't get why that would inhibit bead binding, but it might be true. Either way, I'm glad you are no longer having issues either!

09-22-2014, 04:09 PM	#12
docphil Junior Member Location: Seattle WA Join Date: Aug 2014 Posts: 8	My Lab uses AMPure beads at 0.7x to remove primer-dimer and any junk below our target after barcoding and final pooling for MiSEQ. We elute into TE and then run the eluted DNA through a QiaQuick spin column to remove any residual beads. You can also adjust the amount of EB you spin through the column if you need a higher DNA concentration. Might be a bit redundant but hopefully it helps!