Recently, I have performed PCR reactions to amplify different gene fragments for cloning. I usually included negative controls in my experiments. The negative controls contain all the reaction mixtures except templates for amplification of target genes. Sometimes, I have detected DNA bands on agarose gel from the PCR products of negative controls. Surprisingly, the size of the DNA bands are almost similar to the molecular size of target DNAs. Did any one come across the same problem before? How could it happened?
Seeing bands in your negative control lanes is usually indicative of a contamination. Your gel box should ideally be in a wholly separate room then your thermal cycler (DNA is amazingly robust and can hop jump around on air currents if you're not careful).
It is also possible your pipette is contaminated going into your PCR reaction. clean it out and wipe is down with bleach. Also make certain your tips are filtered.
I'm sure you can think of other sources of contamination.
If it is not contamination, it is also possible that your PCR dimers will form hetero or homodimers with themselves which are often pretty stable. Performing a PCR clean-up (column based assay from a bunch of commercial sources) usually clears that particular problem up.
Good luck!
Seeing bands in your negative control lanes is usually indicative of a contamination. Your gel box should ideally be in a wholly separate room then your thermal cycler (DNA is amazingly robust and can hop jump around on air currents if you're not careful).
It is also possible your pipette is contaminated going into your PCR reaction. clean it out and wipe is down with bleach. Also make certain your tips are filtered.
I'm sure you can think of other sources of contamination.
If it is not contamination, it is also possible that your PCR dimers will form hetero or homodimers with themselves which are often pretty stable. Performing a PCR clean-up (column based assay from a bunch of commercial sources) usually clears that particular problem up.
Good luck!
You have contaminated your PCR reaction. It's good that you are including a negative controls so that you can detect contamination before you keep going with your experiment.
Throw away ALL of your reagents (buffer, water, primers, DNA polymerase, etc.), clean your work space and try agin.
You don't need filter tips if you have good technique. Primer dimers are usually much smaller than your expected product, so you can rule those out.
Make sure you aren't using the same micropipettes to load gels and set up PCR. Amplified PCR products are a VERY common source of contamination.
This is due to contamination. Clean your workspace and equipments with bleach and ethanol and use a new aliquot of reagents to repeat the work. Hopefully you will get better results. Good luck.
I agree with the other comments, I would also add that you should set up your PCR in a different room, or in a flow hood in the room where you are running the agarose gel, as you will get an aerosol when you open the PCR tube and this can be a major source of contamination. In the past I have also had problems with the Milli-Q water, it had algae in it and the primers I was using were picking up the gene from this, so if the problem persists you may want to look at these as well.
Dear All,
Many thanks for your answers and suggestions. I will try next time as per your suggestions.
The amplification may be due to contamination of PCR reagents. Even I have faced similar problems. Before throwing reagents, just give a try to find out the culprit (PCR reagent) due to which you are getting false positive amplification. Use fresh reagents and test one component each from old reagent set for PCR. If you find the culprit, no need to waste other reagents.
The bands seen in the negative control is an indication of contamination. Have a separate room for the mastermix free from DNA. Decontaminate your working space with UV or other means as already suggested. Decontaminate fresh reagents except primers and enzymes with UV light for about 10 minutes if you are using the flow hood and run the PCR again. Keep the negative control closed to avoid contamination. if the problem persists, change the primers and enzymes. I hope this works.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning