I'm doing T7E1 experiment to check CRISPR-Cas9 transfection efficiency. Although i see my PCR products in the true site on the agarose gel, i do not see any band when i do T7E1. It is as if no DNA was loaded on the gel (My T7E1 condition is for 1 hour at 37 C degree). What could be the reason for this?
Thank you very much for answers!
There are some factors which might be cause of not observing any band on gel electrophoresis such as low enzyme activity which means that the T7E1 enzyme might be compromised due improper handling or storage, insufficient DNA concentration, you will need to check the DNA concentration level as well Inorder to identify the gaps and maybe you will need to increase the amount of template DNA in the reaction
We usually see that the overall band intensity goes down quite a bit during the T7 treatment. That might be due to non-specific cleavage. Make sure you don't overdigest and load plenty of product.
Thank all of you very much for answers. I tried to optimize (amount of DNA, T7E1 concentration, incubation time) but i observed overdigestion. Our final decision is to send sanger sequencing.
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