| Component | Amount per reaction (μl) | Final |
|---|---|---|
| 5xPCR buffer | 5 | 1x |
| 5 M Betaine | 5 | 1 M |
| 10 mM dNTPs | 0.5 | 200 nM |
| Template from previous PCR | 3 | |
| H2O | to 25 μl | |
| Polymerase (2U/μl) | 0.5 | 1U |
| 2.5 μM each pDONR221_attb1 + pDONR221_attB2 | 2.5 each | 250 nM each |
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