Cells were lysed in plate format in 50μL QuickExtract DNA Extraction Solution (Lucigen QE09050). Crude lysate was then incubated at 65°C for 20 minutes and 95°C for 20 minutes. Primers were designed with Primer3. PCR amplification was performed using Phusion 2X Master Mix HotStart Flex (New England Biolabs M0536L), 10μM primer pair (see Key resources table , Oligonucleotides), and approximately 100ng template DNA. PCR amplicons were subsequently sent for cleanup and Sanger sequencing. Mutational efficiency was then determined by comparison of non-targeting and gene-targeting sample chromatograms using the TIDE Web Tool (Brinkman et al., 2014 (link)).
Phusion 2x master mix hotstart flex
Manufactured by New England Biolabs
Phusion 2X Master Mix HotStart Flex is a ready-to-use PCR mix containing Phusion DNA polymerase and necessary components for high-fidelity DNA amplification. It includes a hot-start mechanism for enhanced specificity and flexibility.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using phusion 2x master mix hotstart flex
1
Efficient Genome Editing Protocol
2
Efficient Genome Editing Protocol
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Cells were lysed in plate format in 50μL QuickExtract DNA Extraction Solution (Lucigen QE09050). Crude lysate was then incubated at 65°C for 20 minutes and 95°C for 20 minutes. Primers were designed with Primer3. PCR amplification was performed using Phusion 2X Master Mix HotStart Flex (New England Biolabs M0536L), 10μM primer pair (see Key resources table , Oligonucleotides), and approximately 100ng template DNA. PCR amplicons were subsequently sent for cleanup and Sanger sequencing. Mutational efficiency was then determined by comparison of non-targeting and gene-targeting sample chromatograms using the TIDE Web Tool (Brinkman et al., 2014 (link)).
3
Genomic DNA Extraction and Mutation Analysis
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Cells were lysed in plate format in 50µL QuickExtract DNA Extraction Solution (Lucigen QE09050). Crude lysate was then incubated at 65°C for 20 minutes and 95°C for 20 minutes. Primers were designed with Primer3. PCR amplification was performed using Phusion 2X Master Mix HotStart Flex (New England Biolabs M0536L), 10µM primer pair (see Key Resources Table Oligonucleotides), and approximately 100ng template DNA. PCR amplicons were subsequently sent for cleanup and Sanger sequencing. Mutational efficiency was then determined by comparison of non-targeting and gene-targeting sample chromatograms using the TIDE Web Tool (Brinkman et al., 2014) .
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