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?
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?
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