Hi everyone,
I have a specific problem for a few months now with our in-house library preparation for Illumina. We have lower than expected yields by a significant margin - in extreme cases we get 20 ng total out of 10ng of starting material after 18 cycles of PCR, which is ridiculous.
We know that from the very same samples we get great yields with the Illumina kit when done by the core facility (800 ng final).
What steps are the most susceptible to be the cause of the problem?
We use End-it kit for end-repair, followed by A addition with Klenow exo-, ligation of adapters (PAGE purified with a thio bond at the last base) with Quick ligation kit from NEB, gel size selection and PCR with KAPA (Phusion gives the same, so its probably not the PCR step). So everything is pretty standard.
Between every step we cleanup with Qiagen and at the last one with Ampure to get rid of adapter dimers, which we seem to get a LOT of when things are not working right.
To be clear, the protocol gave great results in the past, but at some point yields decreased significantly.
Is it possible that some reagents are more sensitive to freeze / thaw? What would you troubleshoot first? I know the End-it kit isn't directly in cause as results were the same when we got a new one.
I'll try to reanneal adapters from the 100 uM stock to 15 uM working but aside from that I have no idea why it would fail that bad. Please help if you can
I have a specific problem for a few months now with our in-house library preparation for Illumina. We have lower than expected yields by a significant margin - in extreme cases we get 20 ng total out of 10ng of starting material after 18 cycles of PCR, which is ridiculous.
We know that from the very same samples we get great yields with the Illumina kit when done by the core facility (800 ng final).
What steps are the most susceptible to be the cause of the problem?
We use End-it kit for end-repair, followed by A addition with Klenow exo-, ligation of adapters (PAGE purified with a thio bond at the last base) with Quick ligation kit from NEB, gel size selection and PCR with KAPA (Phusion gives the same, so its probably not the PCR step). So everything is pretty standard.
Between every step we cleanup with Qiagen and at the last one with Ampure to get rid of adapter dimers, which we seem to get a LOT of when things are not working right.
To be clear, the protocol gave great results in the past, but at some point yields decreased significantly.
Is it possible that some reagents are more sensitive to freeze / thaw? What would you troubleshoot first? I know the End-it kit isn't directly in cause as results were the same when we got a new one.
I'll try to reanneal adapters from the 100 uM stock to 15 uM working but aside from that I have no idea why it would fail that bad. Please help if you can
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