Dear @Feodora,
It seems to me the problem of low fungal DNA template, as I could see a faint band in R4 lane equivalent to size of your positive control band.
Increasing the DNA template and/or increasing number of cycles during amplification might help.
Thanks,
I agree with you @Amy Johnson regarding number of maximum cycles but atleast she can go till 30, I think. Moreover, gel purification and re-amplification is a good idea, if you just need that band amplified, which she is getting anyways from pure fungal DNA. It seems that is not her requirement, she needs to amplify It from AFM-colonized roots, probably for some screening purpose.
I also agree with Amy Johnson
. There is very little of the dna that you are interested in . Make a nested primer pair just inside the outer pcr primers then amplify with the outer primers as you are doing. Take 1ul of amplified product diluted about 1:100 and amplify again with the internal primers for 15-20 cycles and if there is fungal dna present it should show after the nested pcr. Because the first amplification enriches the target so much the internal primers do not need to be as well designed as they are almost certain to anneal and work in pcr. It is usually only necessary to be careful of primer dimer production for second set design
Feodora Grace
1. Next time, you should leave an empty lane in-between samples and positive control. So you won't confuse that the 'faint' band (pointed out by Yeshveer Singh) is really produced from your sample or 'spill over' from a positive lane next to it.
2. I saw you have a 'Plant' lane in the gel. Is that a negative control using un-infected 'plant roots'? I suggest you to also do a plant root 'internal positive control'. The root 'internal positive control' should give you a band if there is no PCR inhibitors in your isolated DNA sample. If you don't have a band generated from it, then you probably has some kind of PCR inhibitors in your DNA sample. PCR inhibitors can affect your AMF amplification.
It is probably due to low quality of the DNA used. This might have being as a result of poor DNA extraction procedure or the primer of choice is not used. You may also want to check your pcr procedure.
Feodora Grace By the way, the failure of your PCR can be due to 2 reasons-- (1) AMF inoculation was not successful (or too few colonies formed), (2) PCR failed. We have mostly focused on part (2) problem so far.
You should also check part (1) problem. From the paper below, it mentions: " Inoculation with arbuscular mycorrhizal fungi (AMF) may improve plant performance at disturbed sites, but inoculation may also suppress root colonization by native AMF and decrease the diversity of the root-colonizing AMF community. Which AMF you were PCRing? Do you know when is the best time to do the sampling (for PCR) after inoculation?
Article Inoculation effects on root-colonizing arbuscular mycorrhiza...
Feodora Grace < I extracted the gDNA directly from roots. I was thinking of culturing the roots on PDA and only then extract the DNA. >
I am not sure I understood what did you mean by that. What is the logic behind this? Could you elaborate a little?
Feodora Grace Ok, I understand now. Thanks. I think the later approach is a better one ( "they cultured the roots on Trichoderma selective media first (or PDA). Only then they'll pick the mycelium and proceed for gDNA extraction." ), because you will know that Trichoderma mycelium is there.
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