Oct 09, 2022

Public workspacePCR

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l694n1lqe/v1
  • Ana Belem García González1,
  • Jair Alexis Gardea Sáenz1,
  • Irán Alessandra Chaparro Rodríguez1,
  • Georgina Diego1
  • 1Tecnologico de Monterrey Campus Chihuahua
Elena Elizabeth Mercado Flores
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l694n1lqe/v1
Protocol Citation: Ana Belem García González, Jair Alexis Gardea Sáenz, Irán Alessandra Chaparro Rodríguez, Georgina Diego 2022. PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l694n1lqe/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: September 08, 2022
Last Modified: October 09, 2022
Protocol Integer ID: 69748
Keywords: pcr, protocol
Abstract
50 µL volume PCR protocol
Troubleshooting
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.

AB
Nuclease Free Water17 uL
Buffer 10X PCR5 uL
50 mM MgCl21.5 uL
10 mM dNTP’s1 uL
10 uM Forward primer2.5 uL
10 uM Reverse primer2.5 uL
DNA (25 mg/uL)20 uL
Taq Platinum Polymerase0.5 uL
Final Volume50 uL

Perform the next cycle in thermocycler


ABCD
1 CycleInitial activation2 minutes94ºC
35 CyclesDenaturation30 seconds94ºC
35 CyclesAlignment30 seconds50ºC
35 CyclesExtension2 minutes72ºC
1 CycleFinal Extension5 minutes72ºC
MaintenanceMaintenance-4ºC