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Why do I use the primers in the D1-D2 region to amplify the fungus, and using water as a negative control has a weak band that is the same size as the target fragment? When using the ITS primer, using water as a negative control in the ITS region, there is no band. why does this happen?

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    $\begingroup$ Can you add some more information on the regions and also your protocol? What do you do for the PCR and how do you do your controls. $\endgroup$
    – Chris
    Dec 3 '21 at 7:56
  • $\begingroup$ I use the NL-1F and NL-4R primers to amplify the D1-D2 region of fungi, using the Thermal cracking method to obtain the fungi DNA template. Control does't add DNA template. PCR program is 95℃ 5min, 95℃ 30s, 51.5℃ 30s,72℃ 1min,72℃ 5min, 4℃.36 cycles. The electrophoresis result is that the control has a weak band. I use the ITS1-F and ITS4-R primers to amplify the ITS region. Only change the annealing temperature to 55℃。The control doesn't have band. why does this happen? $\endgroup$ Dec 3 '21 at 8:15
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    $\begingroup$ It would be great if you could use the edit function to add this information to the question. To understand this right, if you amplify the D1-D2 region and use water as a negative control you get a weak band, when you do the same blank control on the ITS region (with the ITS primers of course) you get a blank negative control? $\endgroup$
    – Chris
    Dec 3 '21 at 8:43
  • $\begingroup$ yes, i use water as a negative control has a weak band that is the same size as the target fragment, when amplifying the D1-D2 region? When using the ITS primer, using water as a negative control in the ITS region, there is no band. why does this happen? thank you very much. $\endgroup$ Dec 3 '21 at 8:50
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I think you have a contamination problem. Most likely the primer used to amplify the D1-D2 region are contaminated with your DNA. This can happen easily and underscores the importance of proper negative controls.

Since your negative control with another gene is clean, this points against a contamination of your other reagents (dNTPs, water) or even your pipette (yes, this can happen and I have seen this in the lab). Otherwise the negative control of this reaction would show a band as well. I suppose that all reagents except the primers are the same for both reactions, so this clearly points to a contamination of the primers.

This is easy to solve: Make a fresh dilution of your primer working stock - preferrably at another working space where you do not work with your fungal DNA. If possible use filter tips when doing PCR work to avoid cross contamination of samples.

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    $\begingroup$ I don't think you can say where the contamination is and would treat all reagents and pipettors as potentially contaminated. The contamination could be D1-D2 PCR product which would explain the other control being OK. $\endgroup$
    – Michael_A
    Dec 3 '21 at 12:40
  • $\begingroup$ @Michael_A I would agree, if the negative control of the other gene would also show a band. Since it doesn't (and I suppose that all reagents except the primers are the same between these two reactions) this is highly unlikely. $\endgroup$
    – Chris
    Dec 3 '21 at 12:52
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    $\begingroup$ I agree with Chris. You don't want to have handled amplified PCR product at the same workstation (or with the same pipettes) used for setting up reactions or diluting primer stocks. Filter tips are strongly recommended. And I'd also consider decontaminating all of your work stations and pipettes with a diluted bleach solution or something like DNAzap (check pipette user manual for cleaning instruction first to make sure it won't cause damage, but shouldn't). $\endgroup$
    – MikeyC
    Dec 3 '21 at 18:38

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