hi, i recently just did my first PCR assay to characterise fungi species used as biopesticides. i used coniothyrim minitans and trichoderma virens. the reaction was fine, but i had extra bands formed and i don't quite understand what it means or what could have caused it. i have attached the gel image, the extra bands were formed in the trichoderma virens specie.
dear
It is common in case of non-specific primers. I will suggest to check your product size. If your required product size band is present then you should cut this band and purified using Gel purification kit and send it for sequencing.
Best of Luck
if you increase the annealing tm of primer you probably over come this..what others suggested that is also true
Agreed with @Praveen ...increase Tm by 2-4 C or may be template can be reduced...good luck
Agreed with @Praveen ...increase Tm by 2-4 C or may be template can be reduced...good luck
Hi Glory,
As others have suggested, increasing your Tm a little will reduce mismatches. You have good strong product for both species, so you should be able to tolerate some loss of your primary product, if needed. If you have access to a thermal cycler with a gradient function, you can very quickly find the optimal Tm for both species (make sure to run both).
you have to see this video
https://www.youtube.com/watch?v=ZmqqRPISg0g#t=220
if you have a lot of template and the time of elongation is enought you can also get longer lenght molecules
The two clear bands seams that they are one organism but you can again check the product band size of trichoderma and the other one. If it is a positive control the will be fine. From the gel image, the two bands are the same organism but one is contaminated.
Contamination can be from the sample purity, the primer, even handling of the PCR work ( have in mind that you are new for this kind of work) so any simple mistake can happen like amount of primer mix, concentration and amount of template and also the annealing temperature has to be take into consideration.
In general, I can say you did a very nice work and you got very clear band so the only thing you need is to repeat this run again by using very clean pipette, pipette tips, and if possible use new primer mix and template DNA. I hope you will get clear band with such simple steps.
Good luck
Yes, this is due to non specific primer binding. Increase the annealing temp.
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