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-   -   Problems using Agencourt AmPure XP beads. (http://seqanswers.com/forums/showthread.php?t=71989)

Sylv 10-13-2016 04:41 AM

Problems using Agencourt AmPure XP beads.
 
Hi.
I am testing some DNA samples for doing ddRAD sequencing, and I have a problem.
After restriction digest, I purify the reaction using Agencourt Ampure XP beads. When eluting the DNA in water, the beads are stuck to the wells, and very difficult to resuspend.

Have anyone experienced the same problem, and have som ideas as to what causes this? I was thinking the beads have been drying to long, but drying them for shorter time did not make any difference. A pH problem should affect binding of DNA to the beads, and not stick the beads to the wells right??

I hope someone have some advice for me :)

thermophile 10-13-2016 06:00 AM

You could try vortexing, I've never had beads stick so firmly that they wouldn't go back into solution with a quick vortex. I'm not sure its necessary though, the DNA should elute off as long as they are in contact with the water

jdk787 10-13-2016 06:14 AM

The BSA in your digestion will cause your beads to get sticky and clump together. This shouldn't hurt your yield as long as you are able to get them fully resuspended.

adam.geber 10-13-2016 08:34 AM

What volumes are you working with? Diluting your sample may help with clumping produced by BSA or other components of your digest.

How long are you drying your beads for? Have you tried doing additional ethanol washes? If you're vortexing, are you then spinning down your elution volume before placing it on the magnet to prevent droplets from coating the sides of your wells?

As jdk787 said, though, this is only a problem if you have reduced yield or bead carryover.

Sylv 10-13-2016 10:31 PM

Quote:

Originally Posted by adam.geber (Post 199876)
What volumes are you working with? Diluting your sample may help with clumping produced by BSA or other components of your digest.

How long are you drying your beads for? Have you tried doing additional ethanol washes? If you're vortexing, are you then spinning down your elution volume before placing it on the magnet to prevent droplets from coating the sides of your wells?

As jdk787 said, though, this is only a problem if you have reduced yield or bead carryover.

We start out with 500ng DNA in 35Ál, and add 15Ál restriction mastermix so a total of 50Ál. 75Ál beads are then used for purification.
The beads are dried for 5 min. Illumina protocol says at least 15 min, but distributer of the beads says that is way to long.
I don't vortex, but use a shaker for mixing. That has been enough before, but this time I basically had to scrape the beads off with the pipette tips.

BSA in the cutting reaction?...Thanks for making me aware of that.


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