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This protocol introduces site-directed mutations to plasmids using specially
designed primers and high fidelity thermostable DNA polymerases (e.g., Pfu,
TaKaRa Ex Taq). This protocol is efficient for small plasmids (< 10 kb) and
may be applied to large plasmids.
The two primers are complementary to each other and complementary to the plasmid
sequence except for the positions to be mutated. PCR reactions produces new
template with the mutation and the old plasmid template is digested by DpnI because
it is methylated. A number of variants of the original protocol is available for
making insertions and deletions.
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| Figure 1. DpnI-mediated site-directed mutagenesis. |
Figure 2. DpnI-mediated site-directed deletion. |
1. Primer design. Primers are suggested to be perfectly complementary, and
should contain 12-20 bases on either site of the lesion (mutation, deletion or insertion).
In addition, these are PCR primers, thus should be designed to optimize PCR reactions.
See PCR primer design for guidelines.
2. Purification of oligos by polyacrimide gel is suggested.
3. Setup the following PCR reaction:
| 10X PCR Reaction Buffer (Pfu) |
5 ml |
| Forward primer (20 mM, 20 pmol/ml ) |
1 ml |
| Reverse primer (20 mM, 20 pmol/ml ) |
1 ml |
| 10 mM dNTP (mix of 10 mM dATP, dCTP, dGTP, dTTP each) |
1 ml |
| Pfu DNA Polymerase (2.5 units/ml) |
1 ml |
| DNA template (5-50 ng/ml) |
1 ml |
| ddH2O |
40 ml |
| Total volume |
50 ml |
4. Run PCR using the following program:
| Step 1 |
Denaturation 95 °C |
30 seconds |
| Step 2 |
Denaturation 95 °C |
30 seconds |
| Step 3 |
Annealing 55 °C |
1 minute |
| Step 4 |
Extension 68 °C |
2 minutes per kb of plasmid size |
| Step 5 |
Repeat step 2-4 11-17 times |
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| Step 6 |
Storage 4 °C |
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5. Add 1 ml of DpnI and digest at
37 °C for 1 hour.
6. A purification and blunt-end ligation step may be needed for the deletion design shown in Figure 2.
7. Transform E.Coli cells with 1-2 ml of the
digested PCR products by electroporation or high-efficiency competent cells.
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