| Amount | |
| Plasmid concentration (μg/μL) | 1 |
| Plasmid (μg) | 1 |
| Plasmid volume (μL) | 1 |
| FastDigest BbsI | 1 |
| FastAP | 1 |
| 10X FastDigest Buffer | 2 |
| ddH2O | 15 |
| Total (μL) | 20 |
| Number of pairs | 5 |
| Amount per rxn (μL) | Master Mix (μL) | |
| 10X T4 Ligation Buffer | 1 | 5 |
| ddH2O | 6.5 | 32.5 |
| T4 PNK | 0.5 | 2.5 |
| Master Mix Total | 8 | 40 |
| Oligo 1 (100 μM) | 1 | |
| Oligo 2 (100 μM) | 1 | |
| Reaction Total | 10 |
Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).
Put the phosphorylation/annealing reaction in a thermocycler using the following parameters:
| 37°C | 30 min |
| 95°C | 5 min and then ramp down to 25°C at 5°C/min |
Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.
| Amount | Master Mix (μL) | |
| BbsI digested plasmid concentration (ng/μL) | 50 | |
| BbsI digested plasmid (from step 2, ng) | 50 | |
| BbsI digested plasmid (volume) | 1 | 5 |
| 2X Quick Ligase Buffer | 5 | 25 |
| ddH2O | 3 | 15 |
| Master Mix Total (μL) | 9 | 0 |
| Oligo duplex (1:200 dilution) (from step 3) | 1 | |
| Quick Ligase | 1 | |
| Reaction Total (μL) | 11 |
| Amount (μL) | Master Mix (μL) | |
| 10X PlasmidSafe Buffer | 1.5 | 7.5 |
| 10mM ATP | 1.5 | 7.5 |
| ddH2O | 1 | 5 |
| Master Mix Total | 4 | 20 |
| Ligation reaction (step 4) | 11 | |
| Reaction Total | 15 |
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