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.
I reckon that you use too much enzyme in your digestion. Normally, 1 ug of your target DNA could be digested fully in 37 °C by using 10 units of the enzyme for 1 h or more. As you mentioned above, 0.1 ug DNA has been used as the substrate. So 1 unit of the enzyme, I think, is enough for the digestion. 1 unit of the enzyme accounts for 1/125 ul volume. 0.1 ug substrate is too small to be used for the next step, as you will have to extract you digest from agarose gel to be used for ligation. The extraction rate is about 30%. So increase the amount of your substrate in digestion. Try my suggestion to see if it works.
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