Hi Hanh Nguyen

Fungi have 18S and ITS but do not have 16S. On the other hand, bacteria have 16S but do not have 18S or ITS.

If you have a positive amplification for 16S, then you're dealing with bacteria, and therefore you shouldn't be able to amplify ITS. That also means that your positive control (yeast DNA) should not amplify 16S.

If you are getting both 16S and ITS amplification, then your sample is likely to be contaminated.

Good luck!

Marco

Hello!

You don't mention what kind of samples you have. If you have for example environmental samples or animal tissue samples you can find both fungi and bacteria. But 16S successful amplification doesn't mean that you can have equally successful amplification in ITS.

Moreover, your first pair of primers amplifies ITS1 eukaryotic genomic ribosomal region. I am not aware about where the second pair of primers you used targets, but sometimes, depending on the sample, we have successful amplification in ITS1 and not in ITS2 region, and vice versa. So you could try amplifying ITS2 region as well.

Also you could try to use a polymerase that is not hotstart.

Good luck!

Hi Marco Alexandre Guerre,

Thank you so much for your suggestions. I forgot to mentions, I did PCR on DNAs extracted from soils. It supposed to have both bacteria and Fungi. fITS7bF/ITS4NGSR which work for all of my sample is an alternative choice for ITS1F/ITS2 R (used for earth microbiome project). However, for some reasons more than half of my samples failed with ITS1F/ITS2R. My boss insists on using ITS1F/ITS2R. I am so stuck now. I wish someone can give me some suggestion.

Thanks,

Hanh

Hi,

If I understand well, all the samples are of the same kind and all extracted the same way, but still some of them work targeting ITS1 region and some others not?

Maybe you could try diluting those template DNAs that don't work in PCR, for example 1:5, 1:10, 1:20 in order to dilute inhibitors.

Also maybe you could try a different DNA extraction protocol to reduce inhibitors.

Hi Georgia Tarifa,

Thank you so much for your suggestion. You are right. There is definitely some short of inhibition and it seems to impact more on certain primers than others. I am working on developing protocol to extract DNA from soils. We have to extract about 5000 soil samples from diverse location using KingFisher. I tested my protocol for about 200 samples using 16S primers. I worked well, so I thought it is all good until I have to work with ITS1 primers. It is so sad.

I tested a new wash buffer this week. It looks like it might work based on nano drop results but I have to test with PCR to make sure. Oh if you have any suggestion on improve DNA quality for DNA extraction from soil please share with me.

I also diluted the template like what you suggested but for some soil samples, DNA concentrations are very low to start with so I still hope there is a way that I don't need to dilute too much.

Thank you so much again,

Hanh

Did you try to amplify the ITS2 (with the primer ITS3 and ITS4)?

Hello again,

Have you solved your problem?

Although delayed answer, a very good extraction kit for soils (and not only) is PowerSoil DNeasy kit. As far as I have tested, decreases inhibitors and you can gain enough DNA of good quality.