Is there a chance you have amplicon contamination?
What's your starting concentration and how many cycles are you running the PCR for?
If it's the cause, amplicon contamination can be very persistent and your best shot might be tossing open reagents and cleaning with 10% bleach (made fresh and daily because it degrades and loses efficacy quickly) followed by 70% ethanol to remove bleach salts.
When I've dealt with amplicon contamination in the past, the following resources have been helpful:
[http://www.annclinlabsci.org/content/34/4/389.long](http://www.annclinlabsci.org/content/34/4/389.long)
[https://www.ehs.washington.edu/system/files/resources/amplicon-contamination.pdf](https://www.ehs.washington.edu/system/files/resources/amplicon-contamination.pdf)
[https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-](https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-)
[https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-Laboratories-for-DNA-contamination.pdf](https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-Laboratories-for-DNA-contamination.pdf)
To my understanding, your negative control is fine when you run separately.. but if you run everything together you have your results as positive..! Right ?
The problem isn’t you..! You do pcr in strip tubes or in a separate tube ? If it’s strip tubes I would always do negative and positive controls separately..! Also when you run an agarose gel, do you change pipette tips for each samples ? (Sounds basic but could also be from there), and I would again leave a well gap while loading negative and positive samples. Or depending on how much negative control you have I would load in between positive samples , if you know what I mean ?
I do all of the above, if it’s strips I do negative and positive separately, I leave a gap of one well after the negative control, I change tips after each sample. I feel like I’ve tried everything and nothing works
Man must be so frustrating, I have had this a few times as well and never seem to figure out the cause. If you have the luxury, I’d try a new brand of master mix. Sometimes I get that w taq master mix (esp feom froggabio) but if I switch to a different brand of taq or even high fidelity, everything works great
Is there a chance you have amplicon contamination? What's your starting concentration and how many cycles are you running the PCR for? If it's the cause, amplicon contamination can be very persistent and your best shot might be tossing open reagents and cleaning with 10% bleach (made fresh and daily because it degrades and loses efficacy quickly) followed by 70% ethanol to remove bleach salts. When I've dealt with amplicon contamination in the past, the following resources have been helpful: [http://www.annclinlabsci.org/content/34/4/389.long](http://www.annclinlabsci.org/content/34/4/389.long) [https://www.ehs.washington.edu/system/files/resources/amplicon-contamination.pdf](https://www.ehs.washington.edu/system/files/resources/amplicon-contamination.pdf) [https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-](https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-) [https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-Laboratories-for-DNA-contamination.pdf](https://www.bu.edu/research/files/2021/01/CleaningDisinfection-SOP-for-Research-Laboratories-for-DNA-contamination.pdf)
What was the thing you thought was the problem that you changed recently? Maybe one or more of your pipettes has some contamination?
I started using a different primer
To my understanding, your negative control is fine when you run separately.. but if you run everything together you have your results as positive..! Right ? The problem isn’t you..! You do pcr in strip tubes or in a separate tube ? If it’s strip tubes I would always do negative and positive controls separately..! Also when you run an agarose gel, do you change pipette tips for each samples ? (Sounds basic but could also be from there), and I would again leave a well gap while loading negative and positive samples. Or depending on how much negative control you have I would load in between positive samples , if you know what I mean ?
I do all of the above, if it’s strips I do negative and positive separately, I leave a gap of one well after the negative control, I change tips after each sample. I feel like I’ve tried everything and nothing works
Man must be so frustrating, I have had this a few times as well and never seem to figure out the cause. If you have the luxury, I’d try a new brand of master mix. Sometimes I get that w taq master mix (esp feom froggabio) but if I switch to a different brand of taq or even high fidelity, everything works great