I am genotyping transgenic plants for the presence of transgene which is an artificial miRNA construct. I am using miRNA specific primers to genotype the putative transgenic plants which I recovered in Kan media. Every time I am doing the PCR, I am getting a faint band from DNA of a untransformed plant in addition to some clear and strong band detected in some of the samples (please see the picture). My NTC (water control) is clear, i.e, no band. I used different annealing temperature, various cycles (30-35), different concentration of template but getting the same result. As my NTC is not giving any band, can I take a decision on transgenic plants despite having the faint band in the negative template control? I have attached a gel image where
1-17: Putative transgenic plants
-ve1: Non-template control (water+all reagents)
-ve2: Contaminated DNA of a untransformed plant
-ve3: Uncontaminated DNA of a untransformed plant
ve+: Plasmid DNA
Please give your suggestion on this.
Thanks to everyone
Hi,
I think that these bands in the negative control are unspecific PCR products from different loci. Have you compared the DNA concentrations in all the samples? Low amounts of template could explain the faint bands in some of your genotyped lines (1-17). If all DNA concentrations are almost equal, I would assume that you can ignore the faint bands in the controls at the moment. Additionally, you could repeat this genotyping with different primers. Submit some PCR products of positive lines for sequencing to confirm the result.
Increase the annealing temperature or try a touch-down PCR to enhance the specificity of your PCR. Reducing the number of cycles or the elongation time will just hide this signal.
Good luck with your PCR optimization!
My suggestions:
Sequence the product from non-transformed plants with the same PCR primers, see if it produces the miRNA insert sequence or the primers just happen to bind non-specifically somewhere in the plant genome and give rise to the same product size.
Test if the elution / resuspension buffer you are using for DNA extraction is clean.
Also, for reducing non-specific PCR products, I found that shortening the annealing time to 3 sec and denaturation time to 5 sec worked wonders.
I think its good to sequence your product from transformed plants and non-transformed plants first to figure out whats going on then you can able to fix this issue in better way.
lack of specificity of your primers that anneal somewhere in the genome and generate background?
Can you BLAST the miRNA seq with the crop on which you are working. See if there is same region. If you get, locate the position (chromosome number). Do an ePCR with your specif primers and the chromosome you already found to have that region.
Have you the possibility to appreciate by Q-PCR the difference in Ct between positive and negative material ? bands intensity on agarose gels is not a good indicator, especially with very intense saturated bands.
I suggest you to modify your method, the problem may arise because of slight changes in your protocol.
You have contamination. Throw away ALL of your reagents (buffer, water, enzyme, primers, dNTPs, etc.). Clean your workspace and equipment and remake your reagents.
Also, are you separating between your PRE-PCR work space, and then, POST PCR reading and preparation. ? Try not to mix the two. You might have a contamination of the PCR product itself.
it is unclear which NTC you are using water or true NTC's made with reagents only without any sample and/or without primers; it is right that detection applications with high cycles number and no competition are much more exposed to contaminations, and require specific infrastructure to be safe. Have you got internal control in your reaction?
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