Erroneous PCR product due to plasmid DNA contamination - (Sep/15/2008 )
Is it feasible to get an erroneos PCR product due to contamination with plasmid DNA?
I have been getting a similar (not identical) sequences from different new species (not previosly sequenced). I didn´t like this result, for this reason I processed a known species already sequenced...and surprise! I got again a sequence very similar to the previous group (...and of course very different from its published sequence).
I have heard that plasmid DNA is quite resistant and can contaminate the pipettes, some solution or the work area… If this is truth...Can I suppose that I, recurrently, have been amplifying the DNA of the first sample ligated to the vector?
I consider this extremely improbable, because DNA extract from fresh material is present at the time of initiating the PCR and I don't have any product of PCR in my negative control.
Some of you has had a similar problem?
Yes, it is most certainly possible to contaminate pipettes, bench surface, solutions, open tip boxes, etc with plasmid preparations. It does happen.
Stripping down the pipette and soak the barrel in 1M HCl overnight makes the plasmid contamination problem go away. Wiping the bench surface several times with 0.1M HCl also helps.
However your negative control (I am assuming a no template control with water) is blank. So that would probably rule out plasmid contamination (assuming you didn't use any special pipette to add the DNA template, or make the control)
Could the difference seen in the DNA sequence in your known species be caused by polymorphism within the species?
There is some products to get rid of DNA (dna away, dna zap, etc). Clean the pipettes with a detergent, dna cleaner, alcohol or a very dilute acid soln. take off the piece that expelled the tips and clean. After cleaning UV them at least 15 min/side. For PCR use a sterile barrier tips and a pipette set for PCR only.
Check the primers with blast nucleotide (pubmed website).