Hi,
I am wondering if anyone can help with an interesting result I am finding.
During my library prep I consistently perform AMPure cleans at 0.7x sample volume, which I notice removes most fragments below 300 bp.
The issue I am having is after the PCR and following 0.7x AMPure clean to remove all primer products.
My 12 cycle PCR starts with 10 ng of size selected (400-600 bp) DNA.
It is then cleaned with AMPure beads at 0.7x sample volume. As expected the clean-up removes the primers very well and looks similar to a non-amplified control on the gel. However when I quantify my DNA (Qubit 2.0) I am retrieving only 1-2 ng of DNA (80-90% loss)??
If I run the PCR for 20 cycles I notice a large increase in DNA, so my product is amplifying. Any cleaning on the 12 and 20 cycle PCR products visually appears to only remove <300 bp fragments.
Does anyone have any ideas why I would be seeing this loss?
Thanks
I am wondering if anyone can help with an interesting result I am finding.
During my library prep I consistently perform AMPure cleans at 0.7x sample volume, which I notice removes most fragments below 300 bp.
The issue I am having is after the PCR and following 0.7x AMPure clean to remove all primer products.
My 12 cycle PCR starts with 10 ng of size selected (400-600 bp) DNA.
It is then cleaned with AMPure beads at 0.7x sample volume. As expected the clean-up removes the primers very well and looks similar to a non-amplified control on the gel. However when I quantify my DNA (Qubit 2.0) I am retrieving only 1-2 ng of DNA (80-90% loss)??
If I run the PCR for 20 cycles I notice a large increase in DNA, so my product is amplifying. Any cleaning on the 12 and 20 cycle PCR products visually appears to only remove <300 bp fragments.
Does anyone have any ideas why I would be seeing this loss?
Thanks
Comment