Dear Researchers,
I am interested in isolating genomic DNA from field collected Macro-algae (red algae), which has epiphytic on it. problem is both the organisms are Eukaryotic so i am getting trouble, in getting the dna amplification of my interest. Please suggest me some ideas to get rid off of this epiphytes.
In my opinion simplest way is to construct species specific primers, that will amplify only target DNA. Depends from the region of your interest (ITS?), sequence databases (NCBI) could have reference sequences for both species, but not in all cases. It is possible sometimes optimize PCR reaction to get rid from unspecific amplification.
I am doubt whether you can mechanically separate this epiphyte from the surface before DNA isolation, I guess they are closely coalesce. Anyway, you can still have also many endophytes inside.
Dear Angelika mam,
Thanks for sharing your idea mam, I am planning to try with the primer which is used specially in that genus since i have not identified the species...can you suggest me some idea for optimizing PCR reaction...
With Thanks and Regards..
for PCR optimization there are a lot of detailed information available. First measure and
equalize DNA amount (25-50 ng/uL). I would try then gradient PCR from 48-65 degrees & different MgCl2 concentrations (0.5-2.5 mM). Read about touch-down PCR- good method for specificity increase and to obtain large amount of template.
but it depends on your aim. If you need sequence ITS and identify species, you can cut the band from agarose gel and clone it (that step will differentiate and multiplies bands from all amplified species in the sample). if you have clear band in differing size, you can cut it and try to sequence directly. It is valuable to re-amplify purified band to increase PCR amount and specificity for further sequencing.
But if you know that several similar species are present in the sample and optimization will not give the preference to desired one- cloning is the best way to separate them and obtain good quality sequences.
Thank you mam..I am glad to have you in my circle. thanks for the suggestions mam..
i think u should surface sterilization with sodium hypo chloride 0.1% and wash the samples with glass distilled water after u will go to the isolates the DNA from macro algae
what the purpose to isolate the DNA FROM Macro algae?
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