Colony PCR
Ana Belem García González, Jair Alexis Gardea Sáenz, Irán Alessandra Chaparro Rodríguez, Georgina Elisa Diego Macías
Published: 2022-10-12 DOI: 10.17504/protocols.io.dm6gpjwx8gzp/v1
Abstract
Protocol for a PCR from a colony
Steps
1.
Dissolve one colony in 20µL of nuclease-free water and heat at 80°C for 10 minutes.
2.
Prepare the following 50 uL reaction in a 0.2mL PCR tube on ice:
| A | B |
|---|---|
| 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 |
3.
Gently mix the reaction and spin down in the microcentrifuge
4.
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 |
