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.
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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.
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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.
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Originally posted by niceday View PostECO 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.
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.
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