Actually I was checking whether my Primer stock is contaminated or not. So I kept 6 blanks and one positive. Out of 6 blanks I got faint bands in 2 of the blanks(all components except template). All other components except the primer stock were new. What does it mean? How come not all the blanks have the band? Is my Primer stock contaminated? Should I order new Primer stock?
In the gel picture L B B B B B B P L
L- Ladder
B-Blank
P-Positive
Ladder is 100bp and Amplicon size is 428bp
all that you really know is that you have contamination but it may not be in your primer. My feeling is that often contamination comes from the gel running areas when post pcr product gets into the gel and buffer and can get spread everywhere on hands, gloves, pipettes etc. I would clean up your working area and pipettes with bleach and go back to original stock of primers and make up new dilutions. Leave a dish of water on your setup area and just top it up weekly and use a microlitre of it as a test for contamination in the dust and lab atmosphere in every run. If the contamination was on a pipette or splashed on the tubes/strips and dries and sometimes falls into the reaction mix then you can get only occasional amplification in negatives. primers are cheap so probably worth changing but contamination can come from almost anywhere once you have it spreading. Another common route of contamination is aerosol formation when opening tubes/strips when removing the lids can spray tiny droplets of reaction mix everywhere and it sometimes gets on the plasticware.
Sir Rutland,
Thank you sir for quickly responding to my query
This time I actually did PCR and gel run in a different lab where I do not actually work. Thus, I can confidently say that the lab is free of contaminants for the amplicon of my gene of interest.
Thank you again
Patel ma'am,
Thank you for responding to my query within a very short period of time. Your research experience was very helpful.
Dear Anirudha
As Dr Rutland mentioned PCR contamination can come from any one of the components in your reaction. In my experience it can also be associated with the tubes or the pipette tips that are used. This is particualrly the case when you are working with universal primers designed for the amplification of bacterial 16S rDNA or for a variety of eukaryotic markers (both mitochondrial e.g. COI and nuclear 18S). The tubes and tips are prepared using clean room technology but the presence of amplifable DNA (both prokaryotic and eukaryotic) in these disposable plasticwares has been reported by several authors (particularly in the area of metagenomic research). You did not provie details of the target of your assay. Are your primers universal? If so, I recommend that you try a different brand of tube / tip. In my experience this type of contamination occurs at random . Based on your gel, the contamination is very light.... If your PCR is not diagnostic you my be able to live with this level of contamination, or erradicate it by simply reducing the number of cycles in your PCR while increasing the quantity of template DNA. In samples where you have template DNA, the primers will tend to amplify your product rather than the very limited amount of contaminating DNA. This kind of contamination is often just the components of the reaction looking for somthing to do during the PCR reaction..... However, in order to montior this effect, you should make a strong effort to standardize your gels (quantity of agarose per cm3 to ensure that they always the same thickness, control the time of exposure to ethidium bromide or whatever marker substance you use, control the time used for removal of background staining and the exposure time used for capturing images of your gels) and in the short term you should continue to include 4 to 6 controls. This may seem like an unnecessary expense, but it will give you a indication as to both the frequency with which the contamination is occurring and also if the intensity of the contaminating amplicons is increasing over time. Contamination tends to start slowly and then suddenly eveything is strongly positive. Continue to prepare your mastermixes in an different location from that in which you open your tubes post-PCR and where you run your gels. Good luck.
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