After an aliquot of the PCR mixture is analyzed on an agarose gel, the remainder of the reaction is concentrated by ethanol precipitation, resuspended in buffer, and subjected to a simultaneous fill-in/kinase reaction with the Klenow fragment of E. coli RMA polymerase and T4 polynucleotide kinase, the four deoxynucleotides and rATP (34). The reaction then is loaded onto a preparative 1, 1.5, or 2% low-melting temperature agarose gel, depending on the size(s) of the fragment(s) as determined above, and after minimal separation is achieved between the product(s) and the excess primers, the RMA fragments are excised and eluted. After concentration by ethanol precipitation, amplified RMA fragments are ligated into blunt-ended cloning vector, such as SmaI-linearized, dephosphorylated double-stranded M13 replicative form or pUC.

It should be noted that several other methods to purify the RMA fragments for cloning have been investigated. These included standard ethanol-acetate precipitation (1), a 50% ethanol precipitation (35), spin-column purification (36), and precipitation with polyethylene glycol (PEG) (37). The first three methods did not remove sufficient unincorporated primer, and, during the subsequent ligation of the RMA fragment, the primers apparently competed for the blunt ended vector during ligation because the efficiency of ligation was significantly lower and the vast majority of recombinant clones contained only primer derived inserts. Precipitation by polyethylene glycol resulted in only an extremely small RMA pellet, removing the PEG supernatant is difficult, and yields of PCR product were variable.

Protocol

1. Ethanol precipitate the PCR reaction by adding 2.5 volumes of 95% ethanol containing 0.12 M sodium acetate, pH 4.8.

2. Perform the combined fillin-kinase reactions previously described. 3. Add 5 ul of agarose gel loading dye and load the reaction into a well of a 1.0% low-melting temperature agarose gel. Electrophorese for 30-60 minutes at 100-120 mA, and then excise the desired band visualized under UV light with a clean razor blade.

4. Elute the RMA from the gel by standard freeze-thaw methods, followed by a phenol extraction and concentrate by ethanol precipitation.

5. Resuspend the dried RMA in 10 ul of 10:0.1 TE buffer. Use this RMA in a standard blunt-ended ligation reaction. Typically, use 2-3 ul of this RMA in a 10 ul ligation reaction with 20 ng of pUC 18/SmaI-CIAP, although this will depend on the yield of amplified RMA from the PCR reaction.