1. Prepare the single-stranded bacteriophage M13 template as described in Chapter 13, Protocol 1. Purify the uracil-containing template by spun-column chromatography.

2. Phosphorylate the mutagenic oligonucleotide and the universal sequencing primer with bacteriophage T4 polynucleotide kinase. In separate microfuge tubes mix:

synthetic oligonucleotide 100-200 pmoles

10x bacteriophage T4 polynucleotide kinase buffer 2 pl

10 mM ATP 1 pl bacteriophage T4 polynucleotide kinase 4 units

H2O to 20 pl

Incubate the reactions for 1 hour at 37°C and then heat them for 10 minutes at 68°C to inactivate the polynucleotide kinase.

3. Anneal the phosphorylated mutagenic oligonucleotide and universal sequencing primer to the single-stranded bacteriophage M13 DNA containing the target sequence. Mix:

single-stranded template DNA (approx. 1 pg) 0.5 pmole phosphorylated mutagenic oligonucleotide 10 pmoles phosphorylated universal primer 10 pmoles

10x PE1 buffer 1 pl

H2O to 10 pl

Heat the mixture for 5 minutes to 20°C above the theoretical Tm of a perfect hybrid formed by the mutagenic oligonucleotide, calculated from the formula Tm = 4(G+C) + 2(A+T), where (G+C) = the sum of G and C residues in the oligonucleotide and where (A+T) = the sum of the A and T residues in the oligonucleotide. Transfer the tube containing the reaction mixture to a beaker containing H2O at 20°C above the Tm. Stand the beaker on the bench, and allow the reaction to cool slowly to room temperature (approx. 20 minutes). Centrifuge the tube briefly (5 seconds) in a microfuge to collect any fluid that has condensed on the walls of the tube.

4. While the annealing reaction cools to room temperature, mix the following reagents in a fresh 0.5-ml microfuge tube:

10x PE2 buffer 1.0 pl

2 mM dNTP solution 1.0 pl

10 mM ATP 1.0 pl bacteriophage T4 DNA ligase 5 Weiss units Klenow fragment 2.5 units

H2O to 10 pl

Store the mixture on ice until needed.

5. Add 10 pl of the ice-cold reaction mixture from Step 4 to the reaction mixture containing single-stranded DNA and annealed oligonucleotides (Step 3). Incubate the final reaction mixture for 6-15 hours at 16°C.

6. Transfect competent E. coli of an appropriate host strain (e.g., TG1) as follows:

a. Prepare a series of dilutions of the reaction mixture (1:10, 1:100, and 1:500) in 10 mM Tris-Cl (pH 7.6).

b. To a series of chilled (0°C) Falcon 2059 tubes, transfer 1 pl and 5 pl of (i) the original reaction mixture and (ii) each dilution. Add 200 pl of a preparation of competent TG1 cells to each tube.

c. Store the mixtures on ice for 30 minutes, and then transfer them for exactly 2 minutes to a water bath equilibrated at 42°C.

d. Remove the transfected cultures from the water bath, and add 100 pl of a standard overnight culture of TG1 cells. The addition of cells makes it easier to see bacteriophage M13 plaques in the lawn of bacterial cells.

e. Add 2.5 ml of 2x YT top agar (melted and cooled to 47°C) to each tube, and plate the resulting mixtures on separate YT agar plates. Incubate the plates for 12-16 hours at 37°C to allow plaques to form.

7. Screen plaques by sequencing preparations of single-stranded bacteriophage DNA (please see Chapter 12, Protocol 3 or Chapter 12, Protocol 4). If necessary, the plaques can be screened by hybridization with a radiolabeled oligonucleotide probe to detect mutants that arise at a low frequency (Chapter 13, Protocol 7).

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