Hi,
PCR reaction is always mysterious. You never know what you will come up with.
I used to prepare PCR reaction mixture in single appendorf and then shift it to PCR tubes. after shifting I add DNA template.
In my case, my expected PCR product could be a single band at 190 bp [Deletion/deletion (DD)] OR Single band at 490 bp [Insertion/insertion (II)] OR two bands at 190 bp and 490 bp [insertion/deletion (ID)]
But problem here is that
I am observing amplification of exactly same size in Negative control (NC) without DNA template [figure 1] but in similar PCR reaction there is no upper band in Lane 8. what could be the possible reason? If it is giving me amplification in NC then here should be both bands in lane 8 as well.
while in figure 2, I did not run any NC but same reagents, pipette and PCR machine were used to run this reaction. Here why there is no amplification in 3 samples even when I added template in it? If there is contamination in reagents, then there should be amplification in all samples.
Waiting for suggestions and solutions
Thanks,
Misbah
I agree with Jack that there is contamination, You need to run more negative controls to assess the seriousness of the problem otherwise you cannot trust any of your results, You must either get rid of the contaminated reagent/pipettes or move the primers outside of your current set, His rationale of pcr inhibition by something in the dna also looks correct and the inhibition also inhibits the contamination pcr
I also agree with others that there is some contamination from unknown source. The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Be very careful how you open your microcentrifuge tubes. Many people contaminate the inside of lids with their gloves, especially by thumbs on flip caps. Also clean/sterilize the pipette shaft before use.
You can also try to change the working area and choose the one with minimum disturbance or air currents.
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