Large DNA loss post PCR/AMPure clean

[mamor]

Hi,

I am wondering if anyone can help with an interesting result I am finding.

During my library prep I consistently perform AMPure cleans at 0.7x sample volume, which I notice removes most fragments below 300 bp.
The issue I am having is after the PCR and following 0.7x AMPure clean to remove all primer products.

My 12 cycle PCR starts with 10 ng of size selected (400-600 bp) DNA.
It is then cleaned with AMPure beads at 0.7x sample volume. As expected the clean-up removes the primers very well and looks similar to a non-amplified control on the gel. However when I quantify my DNA (Qubit 2.0) I am retrieving only 1-2 ng of DNA (80-90% loss)??

If I run the PCR for 20 cycles I notice a large increase in DNA, so my product is amplifying. Any cleaning on the 12 and 20 cycle PCR products visually appears to only remove <300 bp fragments.

Does anyone have any ideas why I would be seeing this loss?

Thanks

[microgirl123]

Are you using the right concentration of ethanol?

[mamor]

I use freshly made up 70% ethanol for each clean. I am starting to think something may be in the PCR mix that reacts differently with the ampure beads. Otherwise the concentration of 10 ng in 20 uL may to be low - maybe i need to mix throughout the entire 10 minute incubation time.

[microgirl123]

I've been using the PCR cleanup protocol in the Nextera guide with an 80% ethanol wash and it's been working well. Maybe try that?

[mamor]

Thanks I will give it a try.

[addyblanch]

If you're not looking to size select at this point why not up your SPRI bead concentration to the recommended 1.8x? This might help with recovery.

If 0.7x removes fragments ~300bp your product at 400bp might be lost.

Also try washing with 80% Etoh and only dry for 3 minutes.

What are you eluting into? Have you tried MolBio water?

[zatoichi888]

hi, I think you should use 0.9X instead of 0.7X beads. I tried to play around size selection myself and found 0.9X will eliminate your primer dimer etc

[leekb]

hi, I think you should use 0.9X instead of 0.7X beads. I tried to play around size selection myself and found 0.9X will eliminate your primer dimer etc

Ya, I tried 0.7x ampure xp beads that remove not just primer dimers (99bp) but also quite a large amount of my libraries (around 190bp).
So I think 0.9x or 1.0x beads for clean up will be more comfortable.

[zatoichi888]

Ya, I tried 0.7x ampure xp beads that remove not just primer dimers (99bp) but also quite a large amount of my libraries (around 190bp).
So I think 0.9x or 1.0x beads for clean up will be more comfortable.

If you use 1X, you might have to do twice in order to get rid of adaptor dimer etc. Ix cutoff is around 120bp. 0.9X is better to get rid of 120ish bp.