Mar 02, 2022
Colony PCR for screening transgenic Ostreococcus tauri
- 1University of Illinois at Urbana-Champaign
Protocol Citation: Shikha Adhikari, Faith Losbanes, Anbarasu Karthikaichamy 2022. Colony PCR for screening transgenic Ostreococcus tauri. protocols.io https://dx.doi.org/10.17504/protocols.io.b5s4q6gw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 01, 2022
Last Modified: July 11, 2023
Protocol Integer ID: 58940
Keywords: Ostreococcus, colony PCR, PCR, algae, screening
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Abstract
Colony PCR protocol for screening transgenic Ostreococcus tauri
Measure cell concentration using a hemocytometer. Ideally, the cell concentration should be around 1-2x10^5 cells/ml
Mix 50 µL of cells and 50 µL sterile water in a PCR tube.
Boil the PCR tube at 95 °C for 00:05:00 in a thermocycler.
5m
After boiling, briefly centrifuge the PCR tube. Use 1 µL 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.