Contributors: Esther, Chloe, Alexander
epPCR targets
- Project A: Error-prone PCR of the whole CxnA gene
- Project B: Error-prone PCR / site-directed mutagenesis of the Cex (GH10) portion within CxnA
epPCR protocol
| Component | Amount |
|---|---|
| 10× error-prone PCR buffer | 10 µl |
| 50× dNTP mix | 2 µl |
| Extra dNTPs/MgCl₂/MnCl₂ (optional — higher mutation rate) | variable |
| Each primer | 30 pmol |
| Template DNA | 2 fmol (~10 ng of 8 kb plasmid) |
| Taq polymerase (5 U) | 1 µl |
| Water to | 100 µl |
PCR program: 30 s at 94 °C → 30 s annealing → 1 min at 72 °C → 35–50 cycles → 5 min at 72 °C final extension → 4 °C hold.
Quantified product via Nanodrop.
Restriction digest & purification
Digested 5 µg epPCR product with restriction enzymes (200 µl total volume, 37 °C, 6–8 h / overnight). Heat-inactivated, purified via spin columns, eluted in 10 µl pre-warmed water.
Library construction
- Test ligation: 1× ligation buffer + 50 ng vector DNA + T4 ligase (1 U) + insert at 3:1 molar ratio (insert:vector). Control ligation run without insert. Incubated at 16 °C for 12–18 h.
- Transformed into electrocompetent E. coli, plated on LB agar, incubated 37 °C for 12–18 h.
- Colony counts used to estimate final library size (~10³ × test-plate colony count).
Library analysis
Amplified 10–20 random colonies by PCR; ran on gel to confirm amplification; purified and sequenced; aligned against unmutated parental sequence; tabulated point mutations, insertions, deletions, and stop codons to calculate mutation rate and bias spectrum.