I am trying to detect putative transformed tomato plants by PCR using Kanamycin specific primer. Instead of getting one band of 720bp( primer designed for) I am getting multiple bands. I optimized the annealing temperature at 60°C. I am getting single bands in Plasmid DNA( P) no bands in untransformed control tomato(C) and -ve control water(W).
Then I used some other transformed tomato DNA which transformed with stress tolerance gene as other controls; in attached gel pic labelled as S1 and S2; I got single band with this too but when I tested with my transformed tomato plants I have the same problem.
I changed the annealing temp to 63°C but no difference on banding pattern only all bands become very light. Can anyone can help me in this regard? How can I avoid getting these bands? I tried three different primer sets in all I am getting the same problem of multiple banding.
Thanks in advance.
May may want to consider the following:
1. your primers might not be specific therefore one or both of your primers bind to different areas in the gene.
2. there is too much template in the mixture. Usually too much template is the major cause of multiple bands.
* I suggest you try doing the same reaction with only one of the primers for you to determine if you are having problems with primer.
* If you conclude that the primers are both ok than try less temlate.
To me it seems like your primers are amplifying other regions in the template. Do you know whether your primers bind other regions in the genomic DNA? Or could it be possible that your template is contaminated with bacteria DNA? Well, when i had such problems, betaine (Final concentration 1M) helped in getting rid of unspecific bands. Although i was working with human DNA.
One last point, (and im aware that it could be a bit of a more expensive solution), you could try to detect kanamycin with real time PCR using a kanamycin specific Taqman probe in addition to the primers. With this strategy, you would circumvent the detection of unspecific amplicons.
Thanks all friends for your suggestions.
Sinem Karaman first i thought that may be my primer is amplifying other regions as well( DNA is not contaminated with bacteria coz i collected sample from green house after 3 months of transfer) but when i used other tomato DNA( transformed with other plasmid but having kanamycin selection) marked as S1 and S2 in attached gel pic, in that i am getting only single band ; it shows that my primer is designed good as mine test plant is also tomato. But i am not able to figure this out? Does anyone of you have any idea or more suggestions?
OK, another possibility: recently one of my colleagues had such a problem with her regular genotyping PCR. We then figured out that she had solubilized the new DNA in water instead of TE buffer. As you might know EDTA in TE buffer chelates excess Mg2+, => excess Mg increases unspecific bands in PCRs. Is there any chance that you used different buffers for solubilizing your template?
I am not a tomatoe (or eukarytic) guy but so many things can happen during transformation and DNA integration that I can certainly imagine your tomato cells have integrated several copies of the gene and some of these copies went through some rearrangements (deletion or insertion) so that your primers are indeed specific but you have a mix of full length nptII gene, deleted (smaller) and longer gene (integration by transposon or else) so that during PCR you get longer, shorter and normal size bands.
Thanks Robert i was thinking on the same way but not sure about this. If you have any referance article about this, could you please share it with me.Thanks
I have only worked with mammalian DNA/RNA. However, I just took a look at your gel. I have seen a similar banding pattern (just the smaller banding) in my work also.
I found that the problem was in the polymerase, when I changed polymerases/buffers things came up. You may also want to ensure your DNA extraction is really clean. How are you extracting the DNA?
Since I do not know which ladder you are using I am guessing that the top row of banding is about 1400 bp, Is it possible that you are getting double inserts?
I would also like to suggest standardizing your DNA (including controls) before PCRing. I use 25ng/ul - this works for me every single time. (50 ng/15 ul reaction).
Since your S1 and S2 control bands are so weak we cannot tell if the banding pattern existed in them. I would also suggest digesting the amplicons and running digestions and PCRs side by side to see which amplicons were cut. this may give you an idea of what you are dealing with. I hope this is helpful. Best of luck to you and your research.
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