I am doing minicircle cloning, trying to cut with XmaI and EcoRI. It didn't work. I tried several other combination cut such as EcoRI +SalI, BamH1+ EcoRI, it worked just fine. Other combinations such as XmaI+BamH1, SmaI+EcoRI, didn't work. The problem points to the XmaI site. The company did the sequence again, seems the XmaI(SmaI) site is intact. Anybody got any suggestions?
There is one simple method to detect that. Digest your sample with SmaI in one tube and with XmaI in other tube and check them on agarose gel with a uncut minicircle. That will definitely tell you that whether the problem is with your DNA or the enzyme and according to your experiments I would assume the enzyme is not working. So, change the enzyme stock.
Have you also checked the compatibility of these enzymes with the buffer you are using? That also causes problems
I agree with Ethan and Navjyoti.
In addition, make sure that you use Sma I at 25 or 30 °C, not 37!
Maybe your DNA was methylated, thus preventing digestion?
as Ethan mentioned, sequential digestion is the best option. first make sure that your enzymes are working and is cutting the target DNA in ideal reaction conditions and temperature incubation. I also suspect that your enzyme is not working or reaction conditions are not right.
all the best
Sorry I have been away for a long time. Thanks for all your help. My problem has been solved. The issue was that the SYBR Safe I used for staining DNA was not enough ( I switched to SYBR safe from EB), after I added more dye to the running buffer, I was able to see a very weak small band. I have got all my clones. I have tried a lot of different ways to solve the problem. It turned out it was much simpler than I thought.
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