Oct 11, 2022

Public workspaceColony PCR

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpjwx8gzp/v1
  • Georgina Elisa Diego Macías1,
  • Ana Belem García González1,
  • Irán Alessandra Chaparro Rodríguez1,
  • Jair Alexis Gardea Sáenz1
  • 1Tecnologico de Monterrey Campus Chihuahua
Georgina Elisa Diego Macías
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpjwx8gzp/v1
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
Keywords: colony pcr, pcr, protocol
Abstract
Protocol for a PCR from a colony
Troubleshooting
Dissolve one colony in Amount20 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:
ComponentuL Reaction
Magnesium chloride (MgCl2) 25 mM4 µL
5X Colorless GoTaq Buffer5 µL
PCR Nucleotide Mix 10 mM0.5 µL
Forward primer 10 uM1 µL
Reverse primer 10 uM1 µL
GoTaq DNA polymerase (5u/uL)0.2 µL
DNA Colony10 µL
Nuclease-free water3.3 µL
TOTAL25 µL

Gently mix the reaction and spin down in the microcentrifuge
Cycling conditions for a routine PCR:
ABCD
1 CycleInitial activation2 minutes94ºC
35 CyclesDenaturation30 seconds94ºC
Alignment30 seconds50ºC
Extension2 minutes72ºC
1 Cycle72ºC
Maintenance∞4ºC