From 2014.igem.org
Protocols / Exponential Megaprimer PCR (EMP)
The exponential megaprimer PCR (EMP) was used to replace the metal-binding-related sequences of our pORE-E3_2x35S_T4MBP by GFP. In theory, the EMP is based on two PCR reaction: while the insert is amplified by a standard PCR in the first (megaprimer), the second integrates the places the insert in the final vector backbone already. To try different weight ratios of vector backbone and megaprimer turned out be helpful in prior experiments. Since a correct nucleotide sequence was important here, the F-530 Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
Labwork
- Amplify the Megaprimer and dilute the purified product 1:10 before using it for the final EMP reaction.
- Test different weight ratios of Vector:Megaprimer for the best EMP (Table 1).
- Purify the product, dephosphorylate and ligate it.
- Degrade the source vector by DpnI.
- Transfer the EMP product into competent bacteria.
Table 1: EMP Mixes and Temperature Program
Test different ratios of insert and vector DNA amounts. We recommend fours mixes with 10 and 20 ng of insert respectively as well as two mixes lacking the F1 oligonucleotide but contain 80 and 160 ng of insert.
| Volume [μl] | Compounds of standard PCR |
| 4.00 | 5 x Phusion® HF buffer |
| 1.00 | 10 mM µl-1 dNTPs |
| 0.50 | 10 mM F1 oligonucleotide |
| 0.50 | 10 mM µl-1 R2 oligonucleotide |
| ~ | megaprimer |
| 0.20 | Phusion® High-Fidelity DNA Polymerase |
| ~ | template | | ad 20 µl H2O | |
| Cylcer Program | | Step | Temperature [°C] | Time [min] | Cycle no. | | 1. | 98 | 2.0 | 1 | | 2. | 98 | 0.5 | 35 | | 3. | TA | 0.5 | 35 |
| 4. | 72 | - | 35 |
|
|
Table 2: Phosphorylation Mix
To circularize the EMP2 product, the plasmids 5´ends need to be phosphorylated. For that the T4 Polynucleotide kinase (Thermo Scientific) was used as recommended by the supplier.
| Volume [µl] | Compounds |
| 15.0 µl | EMP product |
| 2.0 µl | 10 x Ligase buffer |
| 2.0 µl | 10mM ATP |
| 1.0 µl | Polynucleotide kinase | | | Cylcer Program | | Step | Temperature [°C] | Time [min] | Cycle no. | | 1. | 37 | 10 | 1 | | 2. | 80 | 10 | 1 |
|
Table 3: Ligation
| Volume [µl] | Compounds |
| 1.0 µl | 10mM ATP |
| 1.0 µl | T4 DNA Ligase |
| | Cylcer Program | | Step | Temperature [°C] | Time [min] | Cycle no. | | 1. | 16 | overnight | 1 | | 2. | 80 | 10 | 1 |
|
Table 4: DpnI Digest
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