I'm been trying to sequence these PCR amplified products that are around 550-650 bp long. Extremely pesky to amplify. So far I've tried doing Ampure clean up (at 0.6x) and also gel purification (sybr safe, LMP agarose, etc.). Gel purification does clean up the product very well, but I always end up with just okay recovery.
The SPRI cleanup, on the other hand, gave me much better recovery, and actually cleaned out low mw products <300-350bp quite well. I do still end up with some higher mw minor products > 1k bp, however, that I don't want. Though I think for now, the high mw minor products shouldn't eat up too many of my reads, I would like to get rid of them if possible, without anymore gel purification.
I'm trying to figure out how I would do double spri to get rid of that product.
Something I noticed reading around is that it seems the SPRI bead protocol becomes weird when you try to go below 0.5X beads
roduct ratio; it seems the protocol misbehaves and doesn't have the mw cutoff you would expect (looking at the Broad material slide 61).
I was hoping I could do something like 0.4-0.3X SPRI to retain the 1kb products onto the beads, then take the supernatant and perform the regular 0.6X SPRI to get rid of lower mw products. I also realized, however, that the supernatant taken from the first SPRI cut into the second cut would still have leftover SPRI buffer/PEG. Would I have to change the ratio of the second cut to compensate for the difference?
The SPRI cleanup, on the other hand, gave me much better recovery, and actually cleaned out low mw products <300-350bp quite well. I do still end up with some higher mw minor products > 1k bp, however, that I don't want. Though I think for now, the high mw minor products shouldn't eat up too many of my reads, I would like to get rid of them if possible, without anymore gel purification.
I'm trying to figure out how I would do double spri to get rid of that product.
Something I noticed reading around is that it seems the SPRI bead protocol becomes weird when you try to go below 0.5X beads

I was hoping I could do something like 0.4-0.3X SPRI to retain the 1kb products onto the beads, then take the supernatant and perform the regular 0.6X SPRI to get rid of lower mw products. I also realized, however, that the supernatant taken from the first SPRI cut into the second cut would still have leftover SPRI buffer/PEG. Would I have to change the ratio of the second cut to compensate for the difference?
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