Q5® Hot Start High-Fidelity 2X Master Mix

PCR Hot start PCR - Mammalian DNA

Experiment
PCR Hot start PCR - Mammalian DNA
Product
Q5® Hot Start High-Fidelity 2X Master Mix from New England BioLabs
Manufacturer
New England BioLabs

Protocol tips

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
Always thaw on ice

Publication protocol

General methods

DNA amplification was conducted by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs) unless otherwise noted. All mammalian cell and bacterial plasmids generated in this work were assembled using the USER cloning method as previously described39 and starting material gene templates were synthetically accessed as either bacterial or mammalian codon-optimized gBlock Gene Fragments (Integrated DNA Technologies). All sgRNA expression plasmids were constructed by a 1-piece blunt-end ligation of a PCR product containing a variable 20-nt sequence corresponding to the desired sgRNA targeted site. Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in Supplementary Table 5. All mammalian ABE constructs, sgRNA plasmids and bacterial constructs were transformed and stored as glycerol stocks at −80°C in Mach1 T1R Competent Cells (Thermo Fisher Scientific), which are recA−. Molecular Biology grade, Hyclone water (GE Healthcare Life Sciences) was used in all assays and PCR reactions. All vectors used in evolution experiments and mammalian cell assays were purified using ZympPURE Plasmid Midiprep (Zymo Research Corportion), which includes endotoxin removal. Antibiotics used for either plasmid maintenance or selection during evolution were purchased from Gold Biotechnology.


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Discussion

Discussion

5 years ago

Author: Qatar

When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?

Discussion

5 years ago

Author: Janina Reinhardt Switzerland

Hot start PCR using phusion green DNA polymerase

I am currently performing hot start PCR using phusion green hot start DNA polymerase in order to amplify a 8.8kb insert. However, I cannot get any amplified product at all. I have tried changing some parameters but I am not sure what to do. Any help?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Hot start PCR - Mammalian DNA using Q5® Hot Start High-Fidelity 2X Master Mix from New England BioLabs.

Paper title
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
Full paper
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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Hot Start High-Fidelity 2X Master Mix below.

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