The tricks described below will minimize these effects. This problem has two causes: incomplete digestion of the vector, and re‑ligation of the cut vector with itself. The most common problem with restriction cloning is that the starting vector is recovered after the procedure. Cleaner starting material will yield a better outcome. When in doubt, purify a DNA fragment with a gel.For example, it works better to clone a blunt-ended fragment into a blunt vector site, such as a SmaI site, than into a site that has been blunted with Klenow or T4 DNA polymerase. To maximize efficiency, minimize the number of steps in a procedure.(There is no need to use a spin-column before purifying a DNA fragment with a gel.) Elute the DNA in 40 – 45 μl of 10 mM Tris (pH 8.5), and then perform the next reaction. After each enzymatic reaction, purify the DNA with a spin-column.Start with about 2 μg of DNA when preparing a vector or excising a fragment to be inserted. This approach saves time in the long run. First and foremost, be careful at each step of a procedure.A few simple tricks will help to ensure that your cloning goes smoothly. For many applications, conventional restriction cloning is still the best method.
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