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.
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"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
Someone recently asked me, “how do SPRI beads work” and I realized I was not completely sure so I went to find out. My lab uses kits. Lots...
BW
Roberta
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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.
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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.
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Originally posted by ATϟGC View PostI'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.Attached Files
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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.
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Originally posted by urchin View PostI 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.
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Originally posted by pmiguel View PostHi melop,
What are your elution conditions?
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Phillip
If you want to remove the beads, incubate at 37C overnight.
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