Ampure upper size limit?

[clintp]

Does anyone know if there is an upper size limitation to the DNA that will precipitate and be bound by Ampure XP beads? I'm specifically thinking of cleaning up Phi29 WGA reactions and long cDNAs.

[robertami]

"The size of fragments eluted from the beads (or that bind in the first place) is determined by the concentration of PEG, and this in turn is determined by the mix of DNA and beads. A 50ul DNA sample plus 50ul of beads will give a SPRINA ratio of 1, as will 5ul pipetting (but much harder to get right). As this ratio is changed the length of fragments binding and/or left in solution also changes, the lower the ratio of SPRINA the higher larger the final fragments will be at elution"

Maybe this link can be of help

http://core-genomics.blogspot.co.uk/2012/04/how-do-spri-beads-work.html

BW

Roberta

[clintp]

Thanks for the reply, but that's not exactly what I'm asking. Since the beads are typically used for PCR cleanup and size-selection of small fragments, I wondered if larger molecules of DNA, like 15-100kb, are efficiently bound to the beads and are efficiently eluted.

[robertami]

I've never seen fragments >700 bp with Ampure beads, I guess you could try lowering the Beads: DNA ratio (titrate maybe?) and run the elute on a gel and check the max size you manage to get

[ECO]

HMW products will bind just fine, in fact you'll see the beads clump dramatically with WGA products. You will have problems eluting them so incubate for a while, pipet a bunch and/or vortex.

[clintp]

Very cool--do you actully use them for WGA products?

[melop]

It works for genomic DNA. I now use SPRI beads for extracting genomic DNA either directly from proK-SDS digested tissue or from the supernatant cleaned by phenol-chloroform.

[ATϟGC]

I'll echo ECO and melop in saying that I've tried SPRI beads (Ampure and Seramag speedbeads) to clean up WGA products as well as genomic DNA from home-brew library prep and genomic DNA extraction protocols. As ECO mentioned, they do tend to clump and stick together with higher molecular weight fragments. It might help slightly if you avoid drying the bead pellet till it cracks. Longer elution times (I often incubate overnight at 4 Celsius for genomics extracts) and a small amount of Tween-20 seems to help elute more of the bound DNA.

[melop]

I'll echo ECO and melop in saying that I've tried SPRI beads (Ampure and Seramag speedbeads) to clean up WGA products as well as genomic DNA from home-brew library prep and genomic DNA extraction protocols. As ECO mentioned, they do tend to clump and stick together with higher molecular weight fragments. It might help slightly if you avoid drying the bead pellet till it cracks. Longer elution times (I often incubate overnight at 4 Celsius for genomics extracts) and a small amount of Tween-20 seems to help elute more of the bound DNA.

Yes - I agree. Didn't add tween 20 so recovery is probably lower than optimal, but it's more than enough for what I need so can't complain. Attached is a gel picture of genomic DNA extracted directly after SDS + proteinase K digestion without phenol-chloroform wash from a small piece of fish finclip (1mm x 4 mm). RNA is co-purified because I didn't treat with RNase. Qubit shows a yield of 1.5-2.5 ug per extract.

[urchin]

genomic with spri beads

I know this thread is old but I'm hoping Melop could tell me the ratio of sample to beads used for genomic DNA purification. I looked around online and since the protocols aren't designed for DNA this size I haven't found any suggestions.
Thanks.

[melop]

I know this thread is old but I'm hoping Melop could tell me the ratio of sample to beads used for genomic DNA purification. I looked around online and since the protocols aren't designed for DNA this size I haven't found any suggestions.
Thanks.

You can do as low as 0.6X beads. I usually use 1X. It usually doesn't matter too much for genomic extracts. I also keep the beads in, they don't affect with PCR or library prep.

[pmiguel]

Hi melop,
What are your elution conditions?
--
Phillip

[melop]

Hi melop,
What are your elution conditions?
--
Phillip

I elute in 30ul of Tris pH=8.0. I pipette it until the beads get re-suspended, and directly used that mixture for down-stream workflows without removing the beads.

If you want to remove the beads, incubate at 37C overnight.

[pmiguel]

Ah. So the genomic DNA stays attached to the beads, but still works fine for downstream manipulation. Interesting...

--
Phillip

[snetmcom]

Ah. So the genomic DNA stays attached to the beads, but still works fine for downstream manipulation. Interesting...

--
Phillip

This does work, but the beads will inhibit some reactions.