Sep 25, 2019

Public workspaceQ5 Polymerase PCR Cloning

DOI
dx.doi.org/10.17504/protocols.io.7pqhmmw
  • 1National University of Singapore
NUS iGEM
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DOI: dx.doi.org/10.17504/protocols.io.7pqhmmw
Protocol Citation: NUS iGEM 2019. Q5 Polymerase PCR Cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.7pqhmmw
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 25, 2019
Last Modified: September 12, 2023
Protocol Integer ID: 28112
Materials
MATERIALS
ReagentQ5 High-Fidelity PCR Kit - 200 rxnsNew England BiolabsCatalog #E0555L
STEP MATERIALS
ReagentQ5 Reaction Buffer Pack - 6.0 mlNew England BiolabsCatalog #B9027S
ReagentdNTP Mix, PCR Grade (200 µl)QiagenCatalog #201900
ReagentQ5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
ReagentWater refers to sterilized deionized water
Protocol materials
ReagentQ5 High-Fidelity PCR Kit - 200 rxnsNew England BiolabsCatalog #E0555L
ReagentQ5 Reaction Buffer Pack - 6.0 mlNew England BiolabsCatalog #B9027S
ReagentdNTP Mix, PCR Grade (200 µl)QiagenCatalog #201900
ReagentQ5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
ReagentWater refers to sterilized deionized water
ReagentWater refers to sterilized deionized water
ReagentQ5 Reaction Buffer Pack - 6.0 mlNew England BiolabsCatalog #B9027S
ReagentdNTP Mix, PCR Grade (200 µl)QiagenCatalog #201900
ReagentQ5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
Prepare 6-reaction PCR mix by adding together Amount244.2 µL deionized water, Amount66 µL Q5 reaction buffer, Amount6.6 µL dNTP mix, and Amount3.3 µL Q5 DNA polymerase

ReagentWater refers to sterilized deionized water

ReagentQ5 Reaction Buffer Pack - 6.0 mlNew England BiolabsCatalog #B9027S


ReagentdNTP Mix, PCR Grade (200 µl)QiagenCatalog #201900


ReagentQ5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S

Add Amount1 µL 1 of template DNA and Amount0.5 µL of primer pairs into specified PCR tube

Aliquot Amount48 µL of PCR mix into each PCR tube

Load PCR tubes in thermocycler
Note
Protocol is set to run at Temperature98 °C for Duration00:00:40 followed by Duration00:00:30 for the desired annealing temperature before Temperature72 °C for x amount of time (depending on fragment length). The cycle repeats for 34 rounds starting with Duration00:00:10 atTemperature98 °C followed by annealing and elongation, before incubating at Temperature72 °C for Duration00:05:00 and then Temperature4 °C infinitely. In this PCR run, the elongation time and annealing temperature depends on specific fragment length and primer annealing temperature.