I am having issues resolving PCR inhibition in rhino dung samples for a metagenome sequencing project. I used the DNeasy Blood and Tissue Kit to extract gDNA (with expected low yields, but DNA present all the same), and have attempted to clean extracts amplify the 18S region using NEB's Q5 HiFi 2x Mastermix. I know that the primers themselves are robust, the positive control looks great, and all fecal samples EXCEPT for rhino amplify well! Cleanups/Optimizations that I have tried: 1) Monarch Kit (best Nanodrop 260/230, 260/280 values), 2) Double extractions using Qiagen's DNeasy columns, 3) Dilutions (1:5, 1:10, 1:20, 1:50, 1:75), 4) Optimizing PCR by adding 5% DMSO and 5M Betaine, 5) Zymo's 1-Step PCR Inhibitor Cleanup Kit.
Help!
You can use Guanidine thiocyanate according to Method Genomic DNA extraction from anaerobic digester samples
Try 1:100 dilution and & 5% DMSO at the same time.
Best of luck!
PCR inhibition can be a common issue in fecal samples, especially in wildlife fecal samples, due to the presence of various inhibitors that can co-purify with the DNA during extraction. It seems that you have already tried several approaches to resolve the issue, but still facing the problem.
Here are some additional suggestions that may help you to address the PCR inhibition in your rhino dung samples:
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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