Here's the short description:
We want to ligate adaptors and indexes to 1-3μg of sheared DNA and, in the end, have at least 500ng of adaptor-ligated (and indexed) DNA. It would be great if we could do and still minimize the number of PCR cycles (even just 2 cycles would be great). Does anyone have any advice on how we can do this in the most efficient way possible?
Longer story:
We've been using the NEB Next Ultra DNA kits. We shear 10μg of DNA down to 400bp and size select using beads. At this step we have ~5μg of sheared and size-selected DNA (400bp). We then run it through the NEB DNA Ultra library prep kit using a 6X PCR. After this, in a perfect world (perfect ligation, no loss during cleanups, and 100% efficient PCR), we would expect have 320μg (5 x 2^6) of adaptor ligated and indexes libraries. The problem is that we don't even come close to getting that. In fact, we usually end up with ~200-600ng of adaptor-ligated and indexed DNA.
So my question is: What is going on here? We'd prefer not to use so much starting material and we shouldn't need to. Even if we started with just 1μg and then did 2-4x PCR, we should still have much more than 500ng in our final library. We called NEB and they weren't of much help, so I'm putting this out to the seqanswers community.
Any thoughts?
We want to ligate adaptors and indexes to 1-3μg of sheared DNA and, in the end, have at least 500ng of adaptor-ligated (and indexed) DNA. It would be great if we could do and still minimize the number of PCR cycles (even just 2 cycles would be great). Does anyone have any advice on how we can do this in the most efficient way possible?
Longer story:
We've been using the NEB Next Ultra DNA kits. We shear 10μg of DNA down to 400bp and size select using beads. At this step we have ~5μg of sheared and size-selected DNA (400bp). We then run it through the NEB DNA Ultra library prep kit using a 6X PCR. After this, in a perfect world (perfect ligation, no loss during cleanups, and 100% efficient PCR), we would expect have 320μg (5 x 2^6) of adaptor ligated and indexes libraries. The problem is that we don't even come close to getting that. In fact, we usually end up with ~200-600ng of adaptor-ligated and indexed DNA.
So my question is: What is going on here? We'd prefer not to use so much starting material and we shouldn't need to. Even if we started with just 1μg and then did 2-4x PCR, we should still have much more than 500ng in our final library. We called NEB and they weren't of much help, so I'm putting this out to the seqanswers community.
Any thoughts?
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