During the ligation reaction however the vector can recircularize without the insert DNA. This can be minimized by removing the 5'-phosphate from both termini of the linear vector with Calf Intestinal Alkaline Phosphatase (CIP). Described in the following procedures is a method that has worked consistently. It is recommended to run the vector on a gel cut out the vector band and gel elute to separate the vector from any uncut DNA.The smallest amount of uncut vector will transform efficiently and make finding the bacteria containing the recombinant plasmid virtually impossible. CIP has no effect on uncut circularized plasmids.
A fragment of foreign DNA can be inserted into the vector by a process knows as directional cloning by cutting the vector polylinker with two unique restriction sites. Because of the lack of complementary ends the vector fragment cannot circularize efficiently. The insert DNA however must also have the same cohesive termini as the vector. Directional cloning is widely used when insert DNA orientation is specific. Directional cloning also depletes the number of available cloning sites for further constructing. Specific orientations can be obtained by screening several recombinants from a single-enzyme ligation until the desired orientation is identified.
Another method of ligation though substantially less efficient is blunt-end ligations. Some restriction enzymes cleave both strands of DNA resulting in blunt ends - i.e. no cohesive overhang. Both the insert and vector termini resulting from any restriction enzyme digest can be made blunt. One method utilizes T4 DNA Polymerase and dNTPs to fill in the overhang. Another method uses the enzyme Mung Bean Nuclease to cleave the single stranded overhang. Ligation reactions involving blunt-ended molecules require much higher concentrations of both vector and insert DNA and T4 DNA ligase. One end blunt ligations are slightly more efficient (and directional) and can be achieved by first blunting the linear DNA and then digesting with a restriction enzyme unique to only one end of the DNA. Again both vector and insert must have complementary ends. Either of these methods may change the DNA sequence resulting in the loss of the restriction site.
When it is impossible to find a suitable match between restrictions sites in the plasmid and those at the ends of the insert synthetic linkers can be ligated to the linearized DNA. The procedure requires blunt-end linear DNA fragments to which ligase adds a series of these
synthetic restriction sites. By the digesting with that specific restriction enzyme the resulting DNA has only one linker on each end because the DNA-linker bond is not a restriction site.
Certain restriction overhangs can be modified using Klenow and the recessed 3' termini can be partially filled to generate complementary restriction overhands that are otherwise incompatible. For instance an insert with BamH1 recognition site G'GATC C C CTAG'G has the overhang GATC after digesting. By filling in with dCTP and dTTP and Klenow the resulting overhand is AG. This is compatible with an Acc1 overhang of AG. Note however that the sequence resulting from this ligation is neither a BamH1 site nor an Acc1 site and an alternative method must be used to cut out the insert.
Procedures:
Plasmid Vector Preparation
- Digest 10-20 ug of plasmid DNA with the desired restriction enzyme in a total volume of 100 ul. Incubate appropriately for your specific enzyme (usually 37 degrees C) for 1-4 hours. Supercoiled DNA will take longer to cut and it is recommended to use twice as much enzyme.
- Dephosphorylate the now linear DNA. Add to the completed digest: 30 ul 10X CIP Buffer 167 ul ddH2O and 3 ul CIP enzyme (1 unit per ul). Incubate at 45 degrees C for 25 minutes.
- Prepare an 0.8-1.2% agarose gel with approximately 6 cm wells. This can be achieved by taping several teeth together. Be certain that the taped comb clears the bottom of the gel bed. Also prepare a mini-gel to run a 5 ul aliquot to verifycomplete digestion and determine running time to obtain desired separation.
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