Hi everyone,
I want to amplify soil fungal ITS2 region. But I tried three times with concentrated DNA, diluted DNA, the gel picture always showed double bands. Although I increased the anneal temperature by 2 degrees (from 57 °C to 59 °C), I still got double bands one is about 340 bp, another is 420 bp. Do you think these two bounds may be real products rather than products from non-specifically bound reactions? Can both bands be sequenced by Illumina?
The primers are fITS7 and ITS4.
PCR program is 25 µl reaction: 1 µl of 1:1 diluted DNA + 0.25 µM primers, 25 cycles (95 °C for 30s, 59 °C for 30 s, 72 °C for 30 s)
PCR products were run on 1.5% agarose gel with 100 bp ladder.
Thank you very much for your help.
Xin
When something like this last happened to me, I had a problem with one of my primers causing nonspecific annealing, sequencing both of these bands would be very informative
sequencing these PCR products to get information whether your soil fungal contaminated or not
Length of ITS regions vary within fungal taxa. Therefore, I think it is normal to get multiple bands from soil samples as soil contains a high diversity of fungi.
Illumina (depending your platform) definitely can manage to sequence your read as long as they are overlap (if you do paired end) as the primer pair is designed for metabarcoding (please refer to the original paper). Just be careful when you do the bioinformatic analysis by not trimming the read length to the same length (as normally done on bacterial amplicon sequencing).
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