Hello, I am using KapaHyperPrep Kit for library preparation for Illumina sequencing. I have problem with adaptor dimers formation. Does anyone have some experience with this issue? How did you get rid of them? We tried shorter ligation time and also we used half of the usual amount of adaptors, but there is not that big improvement....
Thank you in advance!
What is the concentration of your input DNA? This can affect the recommended concentration of adaptors you will need in your workflow. Use the table below as a reference:
Input DNA Adapter stock concentration Adapter:insert
molar ratio
1 μg 15 μM 10:1
500 ng 15 μM 20:1
250 ng 15 μM 40:1
100 ng 15 μM 100:1
50 ng 15 μM 200:1
25 ng 7.5 μM 200:1
10 ng 3 μM 200:1
5 ng 1.5 μM 200:1
2.5 ng 750 nM 200:1
1 ng 300 nM 200:1
If you are still having issues, an additional clean up step with beads may be necessary to remove unwanted adaptor dimers (using 0.8X or 1X bead ratio).
Hi Zuzana,
To get rid of adapter dimers, a cleanup step after ligation helps. Also, these can be removed during the size selection step.
Additionally, you can also check the concentration of your PCR/indexing primers.
Please follow the link.
https://support.illumina.com/bulletins/2019/10/what-are-adapter-dimers-.html#:~:text=How%20to%20remove%20adapter%20dimers,may%20reduce%20the%20library%20yields.
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