Editing Error Prone PCR

=== <br>Materials ===

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

=== Method<br> ===

<br>1. Prepare purified plasmid DNA and determine its concentration.<br>2. For each PCR sample, add to tube:<br>10 μL 10X normal error-prone PCR buffer,<br>2 μL 50X dNTP mix,<br>Additional dNTPs (optional),<br>10 μL 55 mM MgCl2 MnCl2 (optional)<br>30 pmol each primer<br>2 fmol template DNA (~10 ng of an 8-kb plasmid)<br>1 μL Taq polymerase (5U)<br>H2O to a final volume of 100 μL.<br>3. Mix sample.<br>4. Place tubes in thermocycler.<br>5. Run Error-Prone PCR Program.<br>30 s at 94°C, 30 s at annealing temperature for primers.<br>1 min at 72°C (for a ~1 kb gene)<br>14–20 cycles,<br>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.<br>7. Purify PCR products either by gel electrophoresis<br>8. Digest with appropriate restriction enzymes. Clean the digested insert, ligate into expression vector, and transform the mutant library into appropriate host strain.<br>9. Grow cultures expressing the mutant library (e.g., in 96-well format) and perform the corresponding assay.

=== <br> ===

=== Notes: <br> ===

<br>1.An appropriate PCR error rate for directed evolution corresponds to a mutation frequency of ~2 to 5 base substitutions per gene.<br>2. Unbalanced dNTP <br> * Increase the amount of dGTP<br> * increase both dCTP and dTTP<br> * increase all but dATP.<br> * Decrease the concentration of certain nucleotides<br> * Decrease the total nucleotide concentration per reaction<br>For example<br>0.2 mM each of dGTP and dATP and 1.0 mM each of dCTP and dTTP.<br>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<br>

[[Category:Protocols]]