PCR
Ana Belem García González, Georgina Diego, Jair Alexis Gardea Sáenz, Irán Alessandra Chaparro Rodríguez
Abstract
50 µL volume PCR protocol
Steps
RECOVERY IDT SEQUENCES
Centrifuge the tubes with the sequences for one minute at 3,000 g x m in the centrifuge
Centrifuge for 5 seconds in the microcentrifuge
Add nuclease-free water needed to have a final concentration of 10 ng/uL.
Vortex, followed by incubating for 20 minutes at 50°C.
Mix briefly by vortex and centrifuge for 8 seconds in mini centrifuge.
PCR
Add nuclease-free water to the first forward and reverse until a concentration of 100 uM is reached, centrifuged quickly in microcentrifuge.
Make dilutions with 10 uL of primers and 90 uL of nuclease-free water to reach a concentration of 10 uM
In 0.2 mL Eppendorf tubes make the following mixture.
| A | B |
|---|---|
| Nuclease Free Water | 17 uL |
| Buffer 10X PCR | 5 uL |
| 50 mM MgCl2 | 1.5 uL |
| 10 mM dNTP’s | 1 uL |
| 10 uM Forward primer | 2.5 uL |
| 10 uM Reverse primer | 2.5 uL |
| DNA (25 mg/uL) | 20 uL |
| Taq Platinum Polymerase | 0.5 uL |
| Final Volume | 50 uL |
Perform the next cycle in thermocycler
| A | B | C | D |
|---|---|---|---|
| 1 Cycle | Initial activation | 2 minutes | 94ºC |
| 35 Cycles | Denaturation | 30 seconds | 94ºC |
| 35 Cycles | Alignment | 30 seconds | 50ºC |
| 35 Cycles | Extension | 2 minutes | 72ºC |
| 1 Cycle | Final Extension | 5 minutes | 72ºC |
| Maintenance | Maintenance | - | 4ºC |
