![]() ![]() Use an excess of restriction enzyme (~20 U for 2 μg DNA), and digest for ~4 hr. Ensure that digestion of the vector goes to completion.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. Restriction Cloning Tipsįor many applications, conventional restriction cloning is still the best method. Sticky ends from different SapI sites may not be compatible.Using a specific example, you will learn to simulate inserting a single fragment into a plasmid vector using restriction and insertion cloning in SnapGene. Sticky ends from different BspQI sites may not be compatible. Sticky ends from different BanII sites may not be compatible.ĪpoI is typically used at 50☌, but is 50% active at 37☌. SmaI can be used at 37☌ for brief incubations. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |