Restriction Enzyme Digestion and Ligation
||1. Choosing restriction enzymes for which the recognition sequences flank your gene of interest
2. Incubate the reaction for the recommended amount of time
3. Purify your fragment
4. Ligate into your plasmid of interest
Agarose Gel Extraction
Most restriction enzymes can be heat inactivated after the digestion is complete however some researchers prefer to purify the DNA fragment before moving into the ligation step. Purification of the fragment occurs by separating the digested fragment of interest on an agarose gel and then the DNA is purified from the agarose.
- Order products for Agarose Gel Extraction
Regardless of the type of end generated by restriction digestion cleavage of the DNA results in fragments with 3’-hydroxyl groups and 5’-phosphate groups. DNA ligase covalently attaches the 5’-phosphate to the 3’-hydroxyl in an ATP dependent reaction, joining two fragments of DNA.
Enzymes for DNA modification
DNA will sometimes need to be modified in order to get successful cloning. Possible modifications can include dephosphorylation (CIAP), filling in of uneven DNA ends (Klenow), etc.