As I said here:
https://www.researchgate.net/post/Looking_for_a_DNA_extraction_protocol_from_adult_beetles_Coleoptera
I finally extracted the DNA from adult beetles using a CTAB protocol. But the DNA is not pure. When I extracted the DNA, the pellet had a brown colour.
Will it inhibit the PCR reaction?
Also you can see the image that I attached (sorry for noise, I took the picture from the CRT-Screen using my cellphone).
I will be grateful for any suggestions to remove the pigments.
Hi, to remove the pigment just use chlorophorm:phenol It should remove all the pigment and organic materials from your samples.
Please find these useful link for protocols:
https://sites.duke.edu/sequencingatduke/files/2013/09/Shared-Protocol-Extracting-DNA-usinig-Phenol-Chloroform.pdf
https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/phenol-chloroform-extraction.html
Hi and thank you Dr. Matny
I don't want to use phenol as it's very toxic and of course I don't have a fume hood in my lab (I must go to the other labs in the Plant Protection department).
I used CTAB without phenol (only with chloroform). Also I didn't use isoamyl too, maybe it helps (adding isoamyl).
I was having problems with fungal pigments inhibiting my PCR reactions.
The EZNA kit by Omega Biotek has a column filtration step and got rid of the pigments completely. It might work for you.
Hi
Thanks Dr. Mayers
But I don't want to buy a kit for this. I can't afford commercial kits.
I did the PCR and everything was OK, I want to send the PCR product for sequencing, I will share the result here.
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