Error Prone PCR

Materials

1. Appropriate PCR amplification primers, designed to have similar melting temperatures, stored at –20°C (see Note 2).
2. Plasmid containing gene of interest to be amplified by mutagenic PCR.
3. 50X dNTP mixture: 10 mM dNTP mix store at –20°C.
4. Individual solutions of dNTPs (10 mM), stored as aliquots at –20°C
5. Taq polymerase. store at –20°C.
6. 10X Normal PCR Buffer: 15 mM MgCl2, 500 mM KCl, 100 mM Tris-HCl, pH 8.3, stored at –20°C.
7. 10X MgCl2 solution: 55 mM prepared in water.
8. 1 mM solution of MnCl2 prepared in water.

Method

1. Prepare purified plasmid DNA and determine its concentration.
2. For each PCR sample, add to tube:
10 μL 10X normal error-prone PCR buffer,
2 μL 50X dNTP mix,
Additional dNTPs (optional),
10 μL 55 mM MgCl2 MnCl2 (optional)
30 pmol each primer
2 fmol template DNA (~10 ng of an 8-kb plasmid)
1 μL Taq polymerase (5U)
H2O to a final volume of 100 μL.
3. Mix sample.
4. Place tubes in thermocycler.
5. Run Error-Prone PCR Program.
30 s at 94°C, 30 s at annealing temperature for primers.
1 min at 72°C (for a ~1 kb gene)
14–20 cycles,
5–10 min at 72°C final extension,

6. Run a sample of the product on a gel to estimate the yield of full-length gene.
7. Purify PCR products either by gel electrophoresis
8. Digest with appropriate restriction enzymes. Clean the digested insert, ligate into expression vector, and transform the mutant library into appropriate host strain.
9. Grow cultures expressing the mutant library (e.g., in 96-well format) and perform the corresponding assay.

Notes:

1.An appropriate PCR error rate for directed evolution corresponds to a mutation frequency of ~2 to 5 base substitutions per gene.
2. Unbalanced dNTP
* Increase the amount of dGTP
* increase both dCTP and dTTP
* increase all but dATP.
* Decrease the concentration of certain nucleotides
* Decrease the total nucleotide concentration per reaction
For example
0.2 mM each of dGTP and dATP and 1.0 mM each of dCTP and dTTP.
This can be prepared using the 50X mixture of dNTPs (10 mM each) followed by addition of individual solutions of 10 mM dCTP and dTTP