JBS Error-Prone Kit

Description: JBS Mutagenesis Series Within three billion years of evolution, nature has produced a plethora of proteins simply by repeated cycles of random mutagenesis followed by in vivo selection for superior function of the encoded proteins.

This example of natural evolution has guided researchers within the last two decades to develop strategies for in vitro permutation of proteins. Among the variety of strategies applied, three major powerful techniques have emerged.

→ JBS dNTP-Mutagenesis Kit #PP-101

The rate of mutagenesis achieved by error-prone PCR is in the range of 0.6-2.0%. → JBS Error-Prone Kit #PP-102

This method allows the recombination of sequences from different, related genes. The overall rate of mutagenesis is approx. 0.7%. → JBS DNA-Shuffling Kit #PP-103

Jena Bioscience now offers all components necessary for each of these techniques 'ready-to-go' in a separate kit, accompanied by a streamlined documentation that maximizes success.

Content

Taq Polymerase (red cap)

5 units/μl, 20 μl

10x Reaction Buffer (blue cap)

10x concentration, 100 μl

10x Error-prone Solution (yellow cap)

10x concentration, 100 μl

dNTP Error-prone Mix (white cap)

unbalanced dNTP ratio (dATP, dCTP, dGTP, dTTP), 40 μl

PCR-grade Water (white cap)

1 ml

For example, proofreading enzymes such as Pfu exhibit error rates in the range from 10-6 to 10-7 whereas non-proofreading enzymes like Taq Polymerase show error rates in the range from 10-4 to 10-5.

This rate however, can be significantly enhanced by modifying the following parameters of a PCR-reaction:

• Higher Mg2+-concentration of up to 7 mM
• Partial substitution of Mg2+ by Mn2+
• Optimized dNTP concentrations at unbalanced rates

Recommended assay preparation

• For a 50 μl reaction, take 5 μl of 10x Reaction Buffer in a sterile vial and refer to Tab. 1.
• Add 2 μl dNTP Error-prone Mix.
• Add template and appropriate primers. Note that depending on the template the required concentration can be up to 10 times higher compared to standard PCR.
• Add 0.4-1 μl Taq Polymerase as recommended.
• Add PCR-grade Water to a final volume of 45 μl.
• Add 5 μl of 10x Error-prone Solution. Note that the solution should be added last to the reaction mixture to prevent precipitation and must be protected from oxidation (oxidation of Mn2+ to Mn3+ may destroy the polymerase).

Tab. 1: Amounts of components for error-prone PCR conditions (50 μl PCR assay)

Component — Amount — Final conc. — Cap

10x Reaction Buffer — 5 μl — 1x — blue

dNTP Error-prone Mix — 2 μl — unbalanced ratio — white

Primers — 20-100 pmol

Template — 3-100 fmol / 2 - 50 ng

Taq Polymerase — 0.4-1 μl — 2-5 units — red

PCR-grade Water — Fill up to 45 μl — white

10x Error-prone Solution — 5 μl — 1x — yellow

Recommended thermocycling conditions

Denaturation — 94°C — 30 sec

Annealing1) — approx. 45-68°C — 30 sec

Extension2) — 72°C — 1 min

Number of cycles: 30 1) The annealing temperature depends on the melting temperature of the primers. 2) The elongation time depends on the length of the fragments to be amplified.

A time of 1 min per kbp is recommended.

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.