- Use multiple channel pipet to pool DNA samples from each column, 10 µl from each well (one mixed sample contains DNA from eight wells). Mix thoroughly.
- Perform nested PCR.
|
PCR Mix |
Stock Concentration |
Amount (µl) |
| H2O | 10.7 | |
| PCR Buffer | 10 X | 1.5 |
| dNTP | 2.5 mM | 1.2 |
| MgCl2 | 50 mM | 0.45 |
| Primers | 25 uM | 0.2 each |
| Taq | 5U/ul | 0.25 |
| Total Volume | 14.5 | |
| DNA Template | 0.5 |
|
PCR Cycles |
|
|
| 94°C | 3 minutes | |
| 94°C | 30 seconds | |
| 55°C | 30 seconds | |
| 72°C | 3 minutes (1 minute for 1kb) | 35 cycles |
| 72°C | 10 minutes |
Use the same nested PCR to screen individual well in the row that the pooled sample shows positive.
We use Platinum Taq (Invitrogen catalog number 10966) for PCR product < 3.5 kb. For PCR product longer than 3.5 kb, we use Elongase (Invitrogen catalog number 10480-010).