![]() You ideally want a recipient plasmid to insert ratio of approximately 1:3. We recommend around 100ng of total DNA in a standard ligation reaction. Once you have cut out and purified your insert and recipient plasmid backbone bands away from the gel via your favorite gel purification method, it is important to determine the concentration of recovered DNA.Ĭonduct a DNA Ligation to fuse your insert to your recipient plasmid. ![]() In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with troubleshooting if your digests don’t look as you expected. Because of this we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples. When running a gel for purification purposes it is important to have nice crisp bands and to have space to cut out the bands. Run your digested DNA on an agarose gel and conduct a gel purification to isolate the DNA. Isolate your insert and vector by gel purification:.CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are commonly used. You should treat your digested recipient plasmid with a phosphatase prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose. If you are going to use only one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent re-circularization of the recipient plasmid. It is also critical that as much of the recipient plasmid as possible be cut with both enzymes, and therefore it is important that the digest go at least 4 hours and as long as overnight. We recommend 1.5-2μg of donor plasmid and 1μg of recipient plasmid. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting material. Set up restriction digests for your donor and recipient plasmids. If you select enzymes that can function in the same buffer, it will save you time in future steps. New England Biolabs for more information about restriction enzyme buffers). If you are lucky enough to have multiple options for enzymes that flank your insert and will result in correct orientation in the recipient plasmid, it is useful to see if one set of enzymes will work in the same restriction enzyme buffer (see ![]() Adding desired restriction sites to your recipient plasmid : You can modify the MCS of your recipient plasmid using Annealed-oligo Cloning.However, you still need to avoid restriction enzymes that cut within your insert. This will allow you to produce a version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS. Adding desired restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends of your oligos.If you cannot find enzymes that meet these criteria, do not fear. It is also possible to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned in the correct orientation. ![]() Ideally, you will find two different restriction enzymes for your subcloning.
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