Oct 11, 2022
Colony PCR
- Georgina Elisa Diego Macías1,
- Ana Belem García González1,
- Irán Alessandra Chaparro Rodríguez1,
- Jair Alexis Gardea Sáenz1
- 1Tecnologico de Monterrey Campus Chihuahua
Protocol Citation: Georgina Elisa Diego Macías, Ana Belem García González, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz 2022. Colony PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjwx8gzp/v1
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: October 11, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 71137
Abstract
Protocol for a PCR from a colony
Dissolve one colony in 20 µL of nuclease-free water and heat at 80°C for 10 minutes.
Prepare the following 50 uL reaction in a 0.2mL PCR tube on ice:
| Component | uL Reaction | |
| Magnesium chloride (MgCl2) 25 mM | 4 µL | |
| 5X Colorless GoTaq Buffer | 5 µL | |
| PCR Nucleotide Mix 10 mM | 0.5 µL | |
| Forward primer 10 uM | 1 µL | |
| Reverse primer 10 uM | 1 µL | |
| GoTaq DNA polymerase (5u/uL) | 0.2 µL | |
| DNA Colony | 10 µL | |
| Nuclease-free water | 3.3 µL | |
| TOTAL | 25 µL |
Gently mix the reaction and spin down in the microcentrifuge
Cycling conditions for a routine PCR:
| A | B | C | D | |
| 1 Cycle | Initial activation | 2 minutes | 94ºC | |
| 35 Cycles | Denaturation | 30 seconds | 94ºC | |
| Alignment | 30 seconds | 50ºC | ||
| Extension | 2 minutes | 72ºC | ||
| 1 Cycle | 72ºC | |||
| Maintenance | ∞ | 4ºC |