SPRI Troubleshooting

teuthbrush · 04-29-2019, 04:40 PM

Hey folks, I'm running into a weird issue with my SPRI cleanups and I was wondering if anyone had any advice.

So, I'm cleaning up a double digest (BSSSIa, MseI) of 100ng of DNA. The DNA itself is in EB buffer.

My reaction volume is 20ul, and generally involves about 10ul of DNA/EB, 2ul of CutSmart, .25ul of each enzyme, and the rest nfH2O. For the cleanup, I'm using homemade SPRI beads, fairly identical to the ones described in the OpenWetware protocol.

Here's the thing: after the ethanol washes, I add water to elute my product, but no amount of pipetting in the world can get my beads to resuspend. Even worse, what does resuspend then immediately gets stuck to the inside of my pipette tips.

If I run a "dummy" digestion, all the same stuff but no enzyme, the beads resuspend as normal, but they simply won't budge if there's been enzyme in the mix. Additionally, I've tried titrating the ethanol drying time down to essentially nothing, and it doesn't help at all. Likewise, I've tried old batches of enzyme, new batches, freshly made beads, trying different ethanol strengths in the wash steps, etc.

If I vortex the everliving crap out of it, I can get most of them off the walls of my plate wells, but I'm still taking a massive hit in yield, in many of my samples.

Has this ever happened to anyone else?

UCan'tBcereus · 04-30-2019, 05:05 AM

Hi teuthbrush,

See the thread below that may interest you:

http://seqanswers.com/forums/showthread.php?t=89063

When you say you did dummy reactions with no enzymes, did you include the Cutsmart buffer? If you did not, it is probable the high levels of BSA (100 ug/mL) in the NEB CutSmart Buffer. Generally it is some contaminant like proteins that will cause the Ampure Beads to clump, but I've seen other people have issues with high salt buffers as well.

If you did include the cutmsmart buffer and did not see clumping, then I am not sure why the enzymes themselves would cause the issue.

teuthbrush · 04-30-2019, 09:43 AM

Quote:

Originally Posted by UCan'tBcereus

Hi teuthbrush,

See the thread below that may interest you:

http://seqanswers.com/forums/showthread.php?t=89063

When you say you did dummy reactions with no enzymes, did you include the Cutsmart buffer? If you did not, it is probable the high levels of BSA (100 ug/mL) in the NEB CutSmart Buffer. Generally it is some contaminant like proteins that will cause the Ampure Beads to clump, but I've seen other people have issues with high salt buffers as well.

If you did include the cutmsmart buffer and did not see clumping, then I am not sure why the enzymes themselves would cause the issue.

I did include the CutSmart buffer, yeah. As far as I know, both the CutSmart and the enzyme itself contain BSA, or at least that was the case last I checked. The thread you linked is this one, by the way. :P

Also bears noting that other steps in my prep (post-ligation SPRI cleanup, post PCR SPRI cleanup) do not have similar clumping issues.

UCan'tBcereus · 04-30-2019, 10:22 AM

Whoops!

The thread I meant to show is:

http://seqanswers.com/forums/showthread.php?t=71989

Besides the BSA (or other residual proteins), the only other thing that I have seen issues with clumping is with large amounts of HMW DNA, which doesn't sound like what's happening to you.

One other way to maybe help would be to try and use DNA LoBind Tubes/Plates and Low Retention tips. I have used both in past when I had bead stickiness issues and it helped a little bit.

teuthbrush · 05-22-2019, 05:49 PM

So, thought I'd update you all.

I tried SPRI again with Agencourt beads, got a slightly better result. But here's the kicker:

As I was trying different things, on one plate, I accidentally grabbed a different plate than the one I was using, where the magnets sit slightly lower (for low elution volumes).

Voila, everything dropped back into solution and resuspended. Somehow, the combination of that magnet and the enzyme caused a situation where it was impossible to resuspend the beads. I noticed that the magnets I used for the time it actually worked made something much more like a ring than the one that didn't work, where it adhered to the walls in something like a smear.

But, hooray, problem fixed. Back to library-making.

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04-29-2019, 04:40 PM	#1
teuthbrush Junior Member Location: California Join Date: Aug 2018 Posts: 9	SPRI Troubleshooting Hey folks, I'm running into a weird issue with my SPRI cleanups and I was wondering if anyone had any advice. So, I'm cleaning up a double digest (BSSSIa, MseI) of 100ng of DNA. The DNA itself is in EB buffer. My reaction volume is 20ul, and generally involves about 10ul of DNA/EB, 2ul of CutSmart, .25ul of each enzyme, and the rest nfH2O. For the cleanup, I'm using homemade SPRI beads, fairly identical to the ones described in the OpenWetware protocol. Here's the thing: after the ethanol washes, I add water to elute my product, but no amount of pipetting in the world can get my beads to resuspend. Even worse, what does resuspend then immediately gets stuck to the inside of my pipette tips. If I run a "dummy" digestion, all the same stuff but no enzyme, the beads resuspend as normal, but they simply won't budge if there's been enzyme in the mix. Additionally, I've tried titrating the ethanol drying time down to essentially nothing, and it doesn't help at all. Likewise, I've tried old batches of enzyme, new batches, freshly made beads, trying different ethanol strengths in the wash steps, etc. If I vortex the everliving crap out of it, I can get most of them off the walls of my plate wells, but I'm still taking a massive hit in yield, in many of my samples. Has this ever happened to anyone else?

04-30-2019, 05:05 AM	#2
UCan'tBcereus Member Location: New England Join Date: Aug 2018 Posts: 12	Hi teuthbrush, See the thread below that may interest you: http://seqanswers.com/forums/showthread.php?t=89063 When you say you did dummy reactions with no enzymes, did you include the Cutsmart buffer? If you did not, it is probable the high levels of BSA (100 ug/mL) in the NEB CutSmart Buffer. Generally it is some contaminant like proteins that will cause the Ampure Beads to clump, but I've seen other people have issues with high salt buffers as well. If you did include the cutmsmart buffer and did not see clumping, then I am not sure why the enzymes themselves would cause the issue.

04-30-2019, 10:22 AM	#4
UCan'tBcereus Member Location: New England Join Date: Aug 2018 Posts: 12	Whoops! The thread I meant to show is: http://seqanswers.com/forums/showthread.php?t=71989 Besides the BSA (or other residual proteins), the only other thing that I have seen issues with clumping is with large amounts of HMW DNA, which doesn't sound like what's happening to you. One other way to maybe help would be to try and use DNA LoBind Tubes/Plates and Low Retention tips. I have used both in past when I had bead stickiness issues and it helped a little bit.

05-22-2019, 05:49 PM	#5
teuthbrush Junior Member Location: California Join Date: Aug 2018 Posts: 9	So, thought I'd update you all. I tried SPRI again with Agencourt beads, got a slightly better result. But here's the kicker: As I was trying different things, on one plate, I accidentally grabbed a different plate than the one I was using, where the magnets sit slightly lower (for low elution volumes). Voila, everything dropped back into solution and resuspended. Somehow, the combination of that magnet and the enzyme caused a situation where it was impossible to resuspend the beads. I noticed that the magnets I used for the time it actually worked made something much more like a ring than the one that didn't work, where it adhered to the walls in something like a smear. But, hooray, problem fixed. Back to library-making.