We are trying to digest a vector with this restriction enzyme. We did not use the oligos, since the vector has two sites. We did not heat it to 65ºC. The vector was extracted using the MaxiPrep Qiagen Kit and appeared in the gel. Also, we used 1 ul of the 125 units Aar1 from Thermo to digest 1 ul of dna in 100ng/ul concentration.
What is exactly the problem, the enzyme does not cut at all or you are having trouble getting it to cut both sites?
Hi Fabio
As mentioned above by Alejandro ..Your query does explains what exactly is the problem ...if your elaborate it ...you'll may get useful responses.
bye
Your question is not very clear. You don't say if the vector is being cut or not. Or are you trying to see if both sites are cut at equally efficiency? Are you trying to ligate a DNA fragment to the restricted vector? One problem you have is that Aar I is a Type I enzyme meaning that it cuts a few nucleotides OUTSIDE the recognition site. You say that the two Aar I sites on your vector are close together. If they are too close, the enzyme may be cutting inside the second adjacent site.
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