Colony PCR for screening transgenic Ostreococcus tauri
Shikha Adhikari, Faith Losbanes, Anbarasu Karthikaichamy
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Abstract
Colony PCR protocol for screening transgenic Ostreococcus tauri
Steps
Measure cell concentration using a hemocytometer. Ideally, the cell concentration should be around 1-2x10^5 cells/ml
Mix 50µL of cells and 50µL sterile water in a PCR tube.
Boil the PCR tube at 95°C for 0h 5m 0s in a thermocycler.
After boiling, briefly centrifuge the PCR tube. Use 1µL from the boiled sample as PCR template.
Mix the PCR components in a sterile laminar hood.
| A | B |
|---|---|
| Component | Volume (uL) |
| 5x green buffer | 5 |
| dNTPs | 0.5 |
| Forward primer | 1.25 |
| Reverse primer | 1.25 |
| Template | 1 |
| GoTaq | 0.125 |
| MgCl2 | 1 |
| H2O | 14.875 |
| Total | 25 |
Perform PCR using the following protocol,
| A | B | C |
|---|---|---|
| Step | Temperature (deg C) | Time |
| Initial Denaturation | 95 | 2 min |
| 30 cycles | 95 | 30 s |
| Ta | 60 s | |
| 72 | 1 min per kb | |
| Final Extension | 72 | 10 min |
| Hold | 4 | HOLD |
Resolve and visualize the PCR product on an agarose gel.
