Hi,
I am working on the T-RFLP and I am trying to optimase this analysis on the ITS regions.
For the restriction enzyme, I use the HaeIII enzyme. I tried this reaction in two different way:
1)10 ng of purified PCR-product plus 10U of enzyme and incubated it at 37°C for 10h or 3h plus 80°C for 20 min;
2)100 ng of purified PCR-product plus 10U of enzyme and incubated it at 37°C for 10 h plus 80°C for 20 min.
In both times, the reaction did not work (I checked the reaction on 3% agarose gel).
Somebody have a idea?
Thanks so much
Giorgia
Hi there,
Two obvious reasons to explain your result:
1)the PCR product doesn't contain any HaeII site
2)HaeII is not functional
Just a quick note : 10U of enzyme for 100ng of DNA is too much as usually 1U is defined as the amount of enzyme required to cut 1µg of DNA in the formatted reaction.
@Dominique Liger thanks so much for your answer.
Tomorrow I will try the reaction with 1 µg DNA and 1U of enzyme (time incubation 2 h at 37°C).
I found other pubblication which use this enzyme on the ITS PCR-product.
I think the main problem might be that you have too little DNA. 10ng of DNA is barely visible on a gel as a single band, if you digest it into multiple bands then you are unlikely to see anything. 100ng should be better but still depending upon the number of bands you expect to see it might also be at the lower limits of detection depending upon your gel and staining and imaging system.
Also 10 hours is much too long and increases the possibility of degradation. I would probably use 5-10 U of enzyme for 1 ug of DNA with a 30-60 min incubation.
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