I am running through some pretty standard library prep for gDNA. Basically, I covaris sheared some zebrafish gDNA, check on BioA (attached). I find that I get a pretty wide distribution of sizes, kinda around 500-600 bp (which is what I'm aiming for). I then used the NEBNext kit to do end repair/dA tailing/Ligation (with custom made y-adaptors using the IDT recommended protocol). I then take all of that, do a SPRI solution at 0.6X to get rid of things that are below 300bp, then I used KAPA hifi to amplify.
Surprisingly, most of the products I get in the amplified PCR product are around 1200-1400bp (also in the attached file), which is much larger than I expected (especially compared to the profile of the sheared DNA template). I don't really know what could've happened.
The products are definitely over amplified (I wanted to make sure I see some product), but these are diverse DNA templates, so I can't really imagine over amplification would give a lot of concatemers (and the peaks don't look like concatemers anyway).
Other thing I could think of is that the sheared DNA was stored at 4 degrees for a couple of days, which I realized isn't best practice, but that should shift the product smaller if anything, not larger.
Another thing may be that the 0.6X SPRI cutoff that i usually use for PCR products may give a different cut off in the presence of the ligation/end repair buffer, but I think this is unlikely. I am running the SPRI cleaned template on bioA too.
I'm a bit confused.
Surprisingly, most of the products I get in the amplified PCR product are around 1200-1400bp (also in the attached file), which is much larger than I expected (especially compared to the profile of the sheared DNA template). I don't really know what could've happened.
The products are definitely over amplified (I wanted to make sure I see some product), but these are diverse DNA templates, so I can't really imagine over amplification would give a lot of concatemers (and the peaks don't look like concatemers anyway).
Other thing I could think of is that the sheared DNA was stored at 4 degrees for a couple of days, which I realized isn't best practice, but that should shift the product smaller if anything, not larger.
Another thing may be that the 0.6X SPRI cutoff that i usually use for PCR products may give a different cut off in the presence of the ligation/end repair buffer, but I think this is unlikely. I am running the SPRI cleaned template on bioA too.
I'm a bit confused.
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