Ampure versus Qiaquick for small amounts of DNA

[Scotch]

Does anyone have any experience using either Ampure or the Qiaquick qiagen columns to clean up small amounts of DNA? I'm only starting with 100ng of DNA and want to lose as little as possible along the way. Which one gives the best DNA recovery? Many thanks in advance.

[ECO]

AMPure. No contest in my experience. Even with MinElute.

[GW_OK]

I'll echo ECO. Ampure is the best DNA purification process going at the moment.

[captainentropy]

I recently make a library (Illumina protocol) from 100ng of ChIPed DNA and I used Qiagen columns and measured the recovery after each step using Qubit. After step 1 (end repair) I recovered almost 100%, after step 2 (A-base addition) about 70%, and after step 3 (adapter ligation) about 75%. However, I use 37 degree buffer EB and elute twice. (so basically I started with 100ng and ended up with a bit over 50ng going into the size selection step).

I've tried Ampure and the recovery was good but took a lot longer. A neighboring lab recently tested Ampure, Ampure XP, and Qiagen and the Ampure XP was the best in their hands. We'll switch to Ampure XP when we run out of the minelute columns.

[SeqR&D]

Just watch your elution volume with the Ampure beads. If you add 90uL of beads, your min amount to elute is ~30uL for best results...you can stretch with 20uL.

[yoon]

Hello,
Can I know when you wash ampure xp beads with 70%. Do you pipet and mix the beads with the etoh? also I was thinking if you can spin the beads down before putting it on the magnet?? Do you guys have any reservations regarding this?

[ECO]

You can resuspend in EtOH but it's main function is salt removal so it's somewhat
overkill.

You can absolutely spin down the beads at any stage, however if you spin hard for longer than about 30 seconds the pellet is difficult to resuspend.

[ECO]

Also as long as we're posting tips...thorough mixing during binding (for at least 2-3 min) is essential for high yields (>85%). A slow rotator is not enough for small volumes.

[zhengz]

Thank you very much for sharing the tips, ECO!

[niceday]

ECO is that 2-3 minutes on a vortex?

I recently eluted some samples after ampure cleanup. Out of curiosity I re-eluted the beads for 3 hours and got 15% DNA back. I'll be speaking to some people to find out if this is the norm.

[SeqR&D]

1. It is recommended to use freshly prepared 80% EtOH.
2. 15 min incubation to bind DNA with multiple vortexing steps during this time period...~10sec per vortex should be fine...just mix things up.
3. It is okay to pipet the beads with the EtOH to mix them (I call this "triterating"), but I usually mix them using the magnet...back and forth. BUT, you can also just add the EtOH and do no mixing at all.
4. As it is recommended to elute 90uL of beads with no less than 30uL, I have sucessfully eluted 160uL of beads with 17uL, with minimal losses...just give it a little extra time.
5. I always spin down my tubes after any mixing/vortexing prior to placing tubes back on the magnet or leaving for further incubation...just a quick spin to get stuff off the tube walls...not compacting the beads.
6. There are better "cleaners/concentrators" than Qiagen...maybe the Zymo...as far as losses go. AMpure XP seems to be the best for yields, but does take much longer. For Qiagen-type column units I tend to bind the DNA twice, and elute twice...same PB/EB, just put it back into the column and spin again. Also, let the EB sit for at least 1min on the filter before spinning.

[youbaosha]

Will switch from Qiagen to Ampure xp!

[austinso]

ECO is that 2-3 minutes on a vortex?

I recently eluted some samples after ampure cleanup. Out of curiosity I re-eluted the beads for 3 hours and got 15% DNA back. I'll be speaking to some people to find out if this is the norm.

Just to add clarification, usually the loss is associated with the "dead volume" (i.e. the liquid between beads) and sometimes the wetting of the plastic surface. So the more beads you use, the greater the dead volume and the poorer your recovery.

In my experience it also depends on how "flattened" the magnetized pellet is, which is why sometimes I use really strong rare-earth magnets for the post wash elution rather than anything on a stand.