Plasmid contamination? From the plasmid preps? Are cultures and preps/isolations done in a different room? If so, do people changing between the prep room and pcr room without changing coat/gloves?

Possibility is maximum for contamination while working, even the tips (very stupid but true fact) the end result always get amplified. Maybe if you could give bit details of the type of the plasmid it can would be better to answer if there is any another cause.

Good Luck

Hi Archita

As mentioned above the amplification of insert even in the control samples or negative template samples indicate contamination ...and it can be in one of the sevral ways right from some PCR reagent to micropipettes. one has to be careful and try to identify the source of conatimation..

bye

You should check the presence of the insert by restriction enzyme cutting and possibly sequence the false insert. It might give you a clue of what exactly you amplify. All the best!!

It sounds like contamination, sorry. Sling away all working solutions and try to find another lab or hood which has never been used for work with your plasmid/gene. Are your primers gene specific or plasmid specific? Trying both can help to find out the source of contamination, the PCR fragment of the gene itself or the plasmid with insert.

The most likely thing is the contamination of other ingredient such as mastermix or primers with your plasmid that can made by an individual error. Use new ingridient or make new working solutions from your stocks and try again. I hope it works.

I strongly feel that to be due to cross-contamination, which can be nullified with the use of filtered tips and maintaining sterile work environment like a PCR-hood. U can directly sequence the amplicon with the respective Primers to know the reason which might be true in ur case too.

The occurrence of false positive results can be due to the following reasons;

(a) Contamination due to accidental usage of same micro-pipette tips for different reagents.

(b) Usage of contaminated reagents (If the reagents are common for several lab members, make your own reagents and use it)

(c) Aerosol contamination, this is due to the repeated usage of same reagents (Primers and template DNA) in a improperly maintained hood, which lead to aerosols of the template DNA to saturate the hood and it will eventually show on your gel as false positive results.

(d) Pipetting error while loading the sample into the wells of the agarose gel.

(e) Breakage in the wells of the agarose gel (Remove the comb only after the addition of the running buffer and remove it smoothly).

(f) Usage of less specific primer pairs.

(g) Usage of wrong template DNA (instead of using plasmid without an insert, one might accidentally use inserted plasmid).

(h) Improper PCR conditions (Use appropriately stringent parameter).

Hope this answers your question.

All the best!

This type of contamination will generally affect every sample in the assay. It occurs when unwanted target DNA is introduced in the assay through e.g. reagents, laboratory disposables, equipment, or the environment

One case we dealt with in the past would indicate possible contamination. We sequenced a template and the results showed not only the wrong result, but the wrong plasmid. As it turned out, the researcher had loaned out the purification kit which was returned with at least one solution contaminated with other plasmid.

If your reagents are used by other people, backwash from pipetting could result in one or more of the PCR reagents being contaminated.

One question I have is how you determine the false positive. Is this a false positive in a negative control?

So cited before, i think that to be due to cross-contamination (strongly), but you can check by sequencing of the obtained fragment ( may be the primers match some where in the plasmid sequence with low percentage and unlikly resulted more or less to the same fragment).

Do you see the false positive signal in a PCR with "no template control" ? If this is the case, you have a contamination problem. This will be highly gene specific, i.e. you have the plasmid with your intended insert somewhere in the lab where it should not be (primer mix, water, pipette tips, pipette, master-mix etc). If you still have the false-positive signals after renewing all PCR compounds, I suggest to go to another lab that has not worked with "your" plasmid before. Plasmids and post-PCR products are the most persistent sources of false-positive PCR or qPCR results. In the case of plasmids or PCR-products, a single molecule (i.e. femtograms) are indeed sufficient to give you a false-positive result. To see this from genomic DNA, you need nanograms.

The best preventive procedure is to have a separate lab for all "dirty" jobs such as plasmid isolation, PCR-electrophoresis and PCR purification. The isolation of DNA, RNA from cells or tissue, however, must be considered "clean" work and kept separate from post-PCR and plasmid methods. The ultimate disaster would be if you get your biological samples (cells, tissues, the DNA from them) contaminated with a plasmid or PCR-product. Because in this worst-case you would not recognise a contamination in your "no-template negative control".

Good luck to get rid of the problem soon.

I agree with other suggestions. The first thing to do is to discard all the reagents including water. Bleach everything, even the bottles that you use for storing water. Clean the bench tops and the shelves with 10% bleach, spay and wipe. Then open new tubes for all the reagents including your primers. Use filtered tips for your experiments.

Another precaution to be taken is never to store cloned DNA such as plasmid in the same refrigerator as the PCR reagents. Genomic DNA should also be stored in a separate refrigerator. Small contamination of the cloned DNA can play an havoc in the lab and most of your PCR reactions will give you unwanted bands.

We have faced this problem before and with above precautions were able to get rid of the problem.

Thank you so much... I will try as suggested... This problem is recurrent in our lab...

I am using all the fresh working stocks, water, aerosol tips, changed the working room areas and further more decontaminated the working area with bleach and UV. Still than consistently i am finding very light intensity of the specified fragment size in the no template control. The forward primer is 17bp and the reverse is 19 bp with custom synthesized Tm of 49 and 51 respectively. Could you pl suggest your experiences for the same.

mr @lakshmi , have you optimized your Tm? ,

Here is another possible reason (This might sound silly, but I saw it very often):  if your PCR amplifies a fraction of a plasmid, you usually get a very, very intense PCR product on the agarose gel (much stronger than from genomic or cDNA PCRs). If on your gel you have an empty well next to the well with the strong plasmid signal, you might frequently have a minimal spill-over of the content from one well to the other. On genomic or cDNA PCRs this usually is not visible (in the empty or no-template lane) , since the signal in the adjacent lane (containing the positive signal) is not to strong.

In the case of a plasmid DNA PCR, however, even a < 5% carry over (~ 0.3 µl) into an empty (or no-template) lane will produce there a visible, weak signal (of exactly the same size . 

I suggest you leave a few lanes empty between your positive plasmid PCR well and the well containing your no-template PCR sample. It is very likely that even in the empty lane next to your positive plasmid PCR a weak band is seen. Only if your no-template sample, loaded a few lanes away now, still shows a weak band, you can consider it a real contamination.

In this case, prepare the no-template PCR in another lab (that has never seen the plasmid), using new primers, new master mix, new water. Work with pipettes that were never used for your plasmid or for post-PCR applications. Never prepare a fresh PCR with pipettes that you used before for gel-loading, PCR purification or for sequencing the plasmid or the PCR product. If you follow these rules, I can not believe that you can still see the false-positive signal.