Hey,

We have experienced same problem in PCR recently and did an extensive troubleshoot for a month. We found  tips and pipettes as culprits, So you must consider this factor.  And I don't think its primer dimer, there is some contaminant for sure.

Based on your description, the most likely reason is still the contamination, because your negative control has no any template but generate weak amplicons with the same size as your target. I suggest that you use all the new reagents including, water, such as UltraPure™ DNase/RNase-Free Distilled Water (Lifetechnologies).

-- What I am asking is can primer dimers form a band around 400bp?

Answer: normally, the sizes of primer dimers are around 100 bp.

Derya,

Primer dimers do not appear as 400bp bands.  You can add the PCR plate to a pre-heated block but do not add reagents to a hot plate or differential evaporaration will be a problem.  Try skipping a lane between your positive and negative control and decrease your PCR cycles by 1-3 and your problem may be solved

Actually, I changed everything (seriously, everything) with new ones but still I am observing the same band. Maybe I should change everything once again. Also while loading I have been definitely very careful and I also tried your suggestion to leave few wells inbetween. Still, there is that little, shiny smiling band. Anyway, I will prepare everything freshly. Thanks for the feedback :)

Are your primers able to amplify human DNA?  It is "floating" through the air.  Also, consider if the manufacturers of the tips and tubes have PCR qualified their products.

No time to read this, maybe it helps: https://www.researchgate.net/post/Problem_with_PCR-large_extra_bands

Are you using filter tips? Your pipette may be contaminated with plasmid DNA for example. You could also try cloning and sequencing the 400bp band to see what it is.

Hi Derya, after you change everything for the second time (don't give up!), I think that it is important to clean very well you pipette, if you can disassemble, clean and assemble again it is even better. I also suggest you to clean your agarose gel apparatus, and all the accessories (as comb, etc), use 1N NaOH, and then wash with H2O. It is really important together with fresh running buffer.

If you can ask some help from other labs, you can ask to prepare your negative control in another room, or ask someone else with different pipette and tips to prepare for you only the negative tubes ih another lab. If you see again the band maybe it is contaminated one of your primer solution stock.

Good luck!

Hi Derya

I think that the problem comes from the agarose gel. Just try to migrate the control alone in a gel  properly prepared.

Thanks for all of the answers. I will definitely consider all of your suggestions including setting up the reaction in a different place and cleaning every apparatus. I think we don't have filter tips but I will also check it out. Thanks a lot.

400bp is too big for primer dimers, and I agree that it might still be contamination. Although you change water etc. it might be that the contamination is actually in your pipettes. I would suggest using filter tips this time to exclude any contamination in your pipettes, as well as using newly opened reagents (H2O, Phusion, buffer, set of primers...).

Def you have contamination in one of your reagents. I suggest you to star with fresh reagent and in a clean environment. Change room as well. Contamination from carry-over are a reality

Hey,

We have experienced same problem in PCR recently and did an extensive troubleshoot for a month. We found  tips and pipettes as culprits, So you must consider this factor.  And I don't think its primer dimer, there is some contaminant for sure.

New primer dilution from the stock solution can also help, or to order new primer. Primerdimers are smaller.

As the others mentioned, consider plastic as well. Don't invest to much time and resources in finding the contaminat. Just clean everything and use fresh reagents (aliquots are nice to circumvent to much loss of material in such cases).

If I see contamination in the negative control, I repeat the PCR with fresh everything (water, reagents, primers, tubes, tips), ideally in a different room.  If you've already done that, another possibility might be carryover from the adjacent well when loading your agarose gel - to eliminate this leave an empty well in between control & samples. 

You can achieve a hot start-like effect simply by preparing the reactions on ice, and not putting them in the PCR machine until it has already heated to 94C.  This helps to reduce mispriming/primer dimer formation, but as others have said your band sounds more like a contaminant. 

I find only two possibilities. Both of them are contaminations in my opinion.

1- It is a contamination

2- You have cross contamination in the agar-gel. Is negative sample close to the other in the gel?

If you want to be totally sure you could digest with restriction enzymes the band. After that, if both have the same pattern, is a contamination, for sure.

Dimers would be much smaller. Primers have been known to self-amplify too, but as the others have said above, such a high molecular weight sounds most like contamination. Make sure you use PCR-grade filter tips (not ones that have been autoclaved) and clean, nuclease-free water. Change your gloves regularly throughout the process. These things should hopefully help! 

Sounds you problem is the pipettes, We had this problem before and dirty pipettes can contaminate your reaction. You can order cleaning for your pipettes and i think your problem will be solved. 

What size is your expected positive control amplicon??

You can sequence amplicon to check it identity!!

If you are doing amplification of human gene - remember - 10% of dust in the room - is human skin!!

Hello everyone, Thanks for all of the valuable recommendations.

Apparently the problem was pipettes. I used completely fresh reagents and done my reaction in another bench somewhere else. It worked, I no more have my band in negative control reaction. One should always keep the pipettes clean :)

Thank you all

Hi, yeah, to have clean pipettes is sometimes hard to fulfill, especially if they are used by other colleagues as well. I strongly recommend filter tips. Thus you can avoid problems with pipettes in the future.

Primer dimer form by-products and decreases your PCR efficiency. It does not form long PCR products (annealing mechanism and amplification as conventional PCR - so you should have a product of 30 - 40 bp, depending on the length of your primers).

So, it seems to be a contamination by your PCR products. Change your tips AND pipettes (do you use filtered tips to prevent aspiration of liquids in pipettes ?). Begin with the negative control and close the tube when you have put all reagents inside.

Hi Derya, In case that you don't have hot start polymerase. You can manually perform the hot-start by suppressing the temperature by preparing the reagents mixing in the ice. You just need to be sure not to let the reaction mixture for  longer at room temperature or higher before it reaches 94 degrees. The thermocycler does warming up slowly and inaccurate amplification could occur at this point. You could avoid this by pause the themocycler once it reached 94 degrees and start to load your PCR tubes in. However the step is not recommended as it will reduce the lifetime of the lid heater.

Hi. I think the primer dimer not related to that case, I ask is the unwanted band just seen in negative control  sample or seen in Other  samples as well?

Primer dimers never exeed 50-70 bp.

400 bp must be amplified contamination.

YOu can check the Tm by real time PCR , must be the same.

discard all reagents, clean bench and pippets and start new..

Hana

Sequence product, do annealing temp gradient PCR