Hi Everyone,
We've been doing a dual-SPRI size selection prior to library prep to try to restrict our fragments to 200-500bp (hoping to sequence PE 100bp on a HiSeq 3000). After this size selection, our fragments typically look like this:
After library prep (using a Kapa LTP library prep kit) our bioanalyzer results typically look something like this:
These are from a 50uL PCR reaction in which we're using 2.5uL of each primer (i5 and i7) at 5uM. We're using 6 cycles of PCR. The input template DNA is 15uL at anywhere from 15 to 50 ng/uL.
Questions:
Thanks very much!
We've been doing a dual-SPRI size selection prior to library prep to try to restrict our fragments to 200-500bp (hoping to sequence PE 100bp on a HiSeq 3000). After this size selection, our fragments typically look like this:
After library prep (using a Kapa LTP library prep kit) our bioanalyzer results typically look something like this:
These are from a 50uL PCR reaction in which we're using 2.5uL of each primer (i5 and i7) at 5uM. We're using 6 cycles of PCR. The input template DNA is 15uL at anywhere from 15 to 50 ng/uL.
Questions:
- Anyone care to speculate what that fat tail of junk is starting at about 600bp? My instinct is that these are fragments where the library fragments are priming each other (overamplification symptoms), but I also think I'm not doing too many cycles and that I probably have enough primer?
- Anyone have any idea if these are suitably sized for a HiSeq 3000/4000?
Thanks very much!
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