PCR inhibitors in DNA?

[grace-ac]

Just ran a qPCR plate targeting V. pectenicida in DNA samples.

The filter sample qPCR plates have been going great, and today was the first time I added in eelgrass swab DNA into the wells.

The efficiency of the run was 130% which google says can mean that there are PCR inhibitors in the templates.

I'm thinking that this is likely only because of the eelgrass swab DNA (the 5 plates I've done of just filter DNA have all had efficiencies 90-110%, which is ideal).

Any thoughts on what I should do? Melanie Prentice mentioned I might want to try a clean up kit on the DNA to get rid of inhibitors.

[grace-ac]

A note - I ran a few swab DNA samples on the nano drop over the weekend and fairly low DNA ng /ul (see post here)... so it's likely not that I have too much DNA loaded in the plate??

[sr320]

a couple of tests

Dilution test. Take 3–4 eelgrass extracts with decent signal, run neat / 1:5 / 1:10. A clean sample shifts ~3.32 Cq per 10× dilution. If the shift is smaller than that, or the 1:10 gives a better-than-expected Cq, you have inhibition.

Spike test. Add a fixed amount of your standard into an eelgrass extract and into water. ΔCq > ~0.5–1 cycle = inhibition, and it works even on samples that are negative for the target.

[grace-ac]

So these tests are suggesting I run a qPCR plate with these different tests (dilution and spike)?

I don't have a ton of supplies to spare.

You don't think I should try the clean-up kit option to remove PCR inhibitors?

[kubu4]

Seeing the actual data would be helpful.

How were your technical reps?

Were your NTCs clean?

How'd the standard curve look?

[sr320]

What he said - link to notebook post…

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On Fri, Aug 14, 2026 at 6:13 PM kubu4 ***@***.***> wrote: *kubu4* left a comment (RobertsLab/resources#2487) <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487*issuecomment-5298683058__;Iw!!K-Hz7m0Vt54!n1MmKsGIT3dOkr2L-YW0KMCtTT8nIHbkFvVvHakMYyJJcv6nFmhP9dOWc_tfZ63Ppk_ym5PrUTS2aQn728fOjyI$> Seeing the actual data would be helpful. How were your technical reps? Were your NTCs clean? How'd the standard curve look? — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487?email_source=notifications&email_token=ABB4PNYFFI767KRPCV4VSOD5J6FJVA5CNFSNUABFM5UWIORPF5TWS5BNNB2WEL2JONZXKZKDN5WW2ZLOOQXTKMRZHA3DQMZQGU4KM4TFMFZW63VHMNXW23LFNZ2KKZLWMVXHJLDGN5XXIZLSL5RWY2LDNM*issuecomment-5298683058__;Iw!!K-Hz7m0Vt54!n1MmKsGIT3dOkr2L-YW0KMCtTT8nIHbkFvVvHakMYyJJcv6nFmhP9dOWc_tfZ63Ppk_ym5PrUTS2aQn759eovek$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ABB4PNYMUZAPU44CWH7XLF35J6FJVAVCNFSNUABEKJSXA33TNF2G64TZHM4TSNRZHA3DQOB3JFZXG5LFHM2TCMZWGA4TAOBXGOQXMAQ__;!!K-Hz7m0Vt54!n1MmKsGIT3dOkr2L-YW0KMCtTT8nIHbkFvVvHakMYyJJcv6nFmhP9dOWc_tfZ63Ppk_ym5PrUTS2aQn7lG-Jn94$> . You are receiving this because you commented.Message ID: ***@***.***>

[grace-ac]

Link to folder with all the exported files from this plate in question: https://drive.google.com/drive/folders/15IxWgCOqXXFz-vXw4p1UcbHvRqRm3MzH?usp=sharing

Notebook post: here

Each sample/standard curve/control was run in triplicate (2ul / well).

All NTCs are clean (0)

[sr320]

That efficiency from standard curve, correct? - what is your standard curve?

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On Fri, Aug 14, 2026 at 6:26 PM Grace Crandall ***@***.***> wrote: *grace-ac* left a comment (RobertsLab/resources#2487) <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487*issuecomment-5298773600__;Iw!!K-Hz7m0Vt54!kaQAUZ59aF_hqBdyaj_B15TP9-Ee92h1nv9XnuxU6GpBEjfoN9ZhGDWRaZXcBePz_11eLWqPkc1HwvkWgRtSXSw$> Link to folder with all the exported files from this plate in question: https://drive.google.com/drive/folders/15IxWgCOqXXFz-vXw4p1UcbHvRqRm3MzH?usp=sharing <https://urldefense.com/v3/__https://drive.google.com/drive/folders/15IxWgCOqXXFz-vXw4p1UcbHvRqRm3MzH?usp=sharing__;!!K-Hz7m0Vt54!kaQAUZ59aF_hqBdyaj_B15TP9-Ee92h1nv9XnuxU6GpBEjfoN9ZhGDWRaZXcBePz_11eLWqPkc1HwvkWqMudOp8$> Notebook post: here <https://urldefense.com/v3/__https://grace-ac.github.io/qPCR-plate4-FHL2026/__;!!K-Hz7m0Vt54!kaQAUZ59aF_hqBdyaj_B15TP9-Ee92h1nv9XnuxU6GpBEjfoN9ZhGDWRaZXcBePz_11eLWqPkc1HwvkWxY1DFag$> Each sample/standard curve/control was run in triplicate (2ul / well). All NTCs are clean (0) Image: Image (view on web) <https://urldefense.com/v3/__https://github.com/user-attachments/assets/27be5602-3399-4885-a166-9d4ba068250e__;!!K-Hz7m0Vt54!kaQAUZ59aF_hqBdyaj_B15TP9-Ee92h1nv9XnuxU6GpBEjfoN9ZhGDWRaZXcBePz_11eLWqPkc1HwvkW-Nskfqo$> — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487?email_source=notifications&email_token=ABB4PNYVL5DE4JBBOAPBI235J6GZPA5CNFSNUABFM5UWIORPF5TWS5BNNB2WEL2JONZXKZKDN5WW2ZLOOQXTKMRZHA3TOMZWGAYKM4TFMFZW63VHMNXW23LFNZ2KKZLWMVXHJLDGN5XXIZLSL5RWY2LDNM*issuecomment-5298773600__;Iw!!K-Hz7m0Vt54!kaQAUZ59aF_hqBdyaj_B15TP9-Ee92h1nv9XnuxU6GpBEjfoN9ZhGDWRaZXcBePz_11eLWqPkc1HwvkWwPWqBWk$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ABB4PNZITQPC3CD74EACNYL5J6GZPAVCNFSNUABEKJSXA33TNF2G64TZHM4TSNRZHA3DQOB3JFZXG5LFHM2TCMZWGA4TAOBXGOQXMAQ__;!!K-Hz7m0Vt54!kaQAUZ59aF_hqBdyaj_B15TP9-Ee92h1nv9XnuxU6GpBEjfoN9ZhGDWRaZXcBePz_11eLWqPkc1HwvkWQFDY0gw$> . You are receiving this because you commented.Message ID: ***@***.***>

[grace-ac]

Yes

Here's the spreadsheet I used to create the standard curve dilutions: https://docs.google.com/spreadsheets/d/1KVyNMiGuKE1-hX4hHQHH0COr6q02rdxb/edit?usp=sharing&ouid=109180173241522173082&rtpof=true&sd=true

[sr320]

So has nothing to do with eelgrass samples? In short I am not seeing what issues is. More holistically I am presuming the standard curve was made once and used on all plates?

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On Fri, Aug 14, 2026 at 6:51 PM Grace Crandall ***@***.***> wrote: *grace-ac* left a comment (RobertsLab/resources#2487) <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487*issuecomment-5298932866__;Iw!!K-Hz7m0Vt54!h3tWWKyLu5ZUQFXN9HlMi1kpIbiVhY9uUq7gS_rkaH2n2baasBViZ3YieTM2geJHfkYbARhNTSTFJPdwaLOLnzk$> Yes Here's the spreadsheet I used to create the standard curve dilutions: https://docs.google.com/spreadsheets/d/1KVyNMiGuKE1-hX4hHQHH0COr6q02rdxb/edit?usp=sharing&ouid=109180173241522173082&rtpof=true&sd=true <https://urldefense.com/v3/__https://docs.google.com/spreadsheets/d/1KVyNMiGuKE1-hX4hHQHH0COr6q02rdxb/edit?usp=sharing&ouid=109180173241522173082&rtpof=true&sd=true__;!!K-Hz7m0Vt54!h3tWWKyLu5ZUQFXN9HlMi1kpIbiVhY9uUq7gS_rkaH2n2baasBViZ3YieTM2geJHfkYbARhNTSTFJPdw1A_znSM$> — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487?email_source=notifications&email_token=ABB4PNYNW4ZBRSUMJNTI2RD5J6JWPA5CNFSNUABFM5UWIORPF5TWS5BNNB2WEL2JONZXKZKDN5WW2ZLOOQXTKMRZHA4TGMRYGY3KM4TFMFZW63VHMNXW23LFNZ2KKZLWMVXHJLDGN5XXIZLSL5RWY2LDNM*issuecomment-5298932866__;Iw!!K-Hz7m0Vt54!h3tWWKyLu5ZUQFXN9HlMi1kpIbiVhY9uUq7gS_rkaH2n2baasBViZ3YieTM2geJHfkYbARhNTSTFJPdwElGhHPU$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ABB4PN6JDUZ5CMAVWV43DI35J6JWPAVCNFSNUABEKJSXA33TNF2G64TZHM4TSNRZHA3DQOB3JFZXG5LFHM2TCMZWGA4TAOBXGOQXMAQ__;!!K-Hz7m0Vt54!h3tWWKyLu5ZUQFXN9HlMi1kpIbiVhY9uUq7gS_rkaH2n2baasBViZ3YieTM2geJHfkYbARhNTSTFJPdwqhzZp24$> . You are receiving this because you commented.Message ID: ***@***.***>

[kubu4]

Spreadsheet link didn't work - says "Page not found"

[kubu4]

Also, notebook entry is lacking info.

What are the standard curves (e.g. plasmids? general sample dilutions?)?

What solution was used for dilutions?

What was starting stock concentration?

How did you quantify the starting stock (e.g. NanoDrop? Qubit?)?

What were your reaction components/volumes?

[kubu4]

Link to folder with all the exported files from this plate in question

Also need raw .pcrd file, please.

[sr320]

I think you will also find it useful to plot data points that contribute to efficiency values

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On Fri, Aug 14, 2026 at 7:13 PM kubu4 ***@***.***> wrote: *kubu4* left a comment (RobertsLab/resources#2487) <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487*issuecomment-5299065284__;Iw!!K-Hz7m0Vt54!kBoPGRjxvHlTclQmCEuJXqrid18VSdsEm2KnpNBJyxhVjlJk49lqWsER-y8whXRikOo39486e9JWHgRANFuV4zM$> Link to folder with all the exported files from this plate in question Also need raw .pcrd file, please. — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2487?email_source=notifications&email_token=ABB4PNZ4VE42ZB7YORFAO6L5J6MIFA5CNFSNUABFM5UWIORPF5TWS5BNNB2WEL2JONZXKZKDN5WW2ZLOOQXTKMRZHEYDMNJSHA2KM4TFMFZW63VHMNXW23LFNZ2KKZLWMVXHJLDGN5XXIZLSL5RWY2LDNM*issuecomment-5299065284__;Iw!!K-Hz7m0Vt54!kBoPGRjxvHlTclQmCEuJXqrid18VSdsEm2KnpNBJyxhVjlJk49lqWsER-y8whXRikOo39486e9JWHgRA8VFHaRE$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ABB4PN67UCXGXB4JLJ76QUD5J6MIFAVCNFSNUABEKJSXA33TNF2G64TZHM4TSNRZHA3DQOB3JFZXG5LFHM2TCMZWGA4TAOBXGOQXMAQ__;!!K-Hz7m0Vt54!kBoPGRjxvHlTclQmCEuJXqrid18VSdsEm2KnpNBJyxhVjlJk49lqWsER-y8whXRikOo39486e9JWHgRAmw0nDj0$> . You are receiving this because you commented.Message ID: ***@***.***>

[kubu4]

Found .pcrd file here: https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/grace_crandall_2026-08-12%2014-02-56_Connect-FHL2026-filters-eelgrassswabs-plate4.pcrd

Looking at it, the baseline threshold is artificially low. When adjusting the baseline manually to a more appropriate level (based on the standards' amplification), the efficiency gets into the acceptable range.

However, your two most dilute set of standards are wonky.

Here's how things look with an adjusted baseline (I've turned the standards green for easier viewing):

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You also have a high number of samples with poor technical reps (and poor amplification - this is driving the auto-calculate baseline too low).

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Basically, if you haven't repeated this yet, then that's the very first thing to do (IMO).

Gotta get those reps tighter.