In a 20 μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5 μM). Approximately 50 ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18 (link)]. Thermal cycler settings included an initial denaturation of 95 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 10 min. The amplicons were assessed using 2% Agarose gel (SeaKem® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure® PCR product purification kit (Roche Molecular Diagnostics, Switzerland).
Emerald gt pcr master mix
Manufactured by Takara Bio
Sourced in United States
The 1X Emerald GT PCR master mix is a ready-to-use solution that contains all the necessary components for performing polymerase chain reaction (PCR) amplification. It includes a proprietary DNA polymerase, dNTPs, and optimized buffer system.
Automatically generated - may contain errors
4 protocols using emerald gt pcr master mix
1
EGFR Gene Mutation Detection Protocol
2
Characterization of EGFR Exons 18-21
Check if the same lab product or an alternative is used in the 5 most similar protocols
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In a 20μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5μM). Approximately 50ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18] . Thermal cycler settings included an initial denaturation of 95°C for 15 minutes, followed by 45 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, extension at 72°C for 45 seconds and a nal extension at 72°C for 10 minutes. The amplicons were assessed using 2% Agarose gel (SeaKem ® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure ® PCR product puri cation kit (Roche Molecular Diagnostics, Switzerland).
3
Characterization of EGFR Exons 18-21
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In a 20μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5μM). Approximately 50ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18] . Thermal cycler settings included an initial denaturation of 95°C for 15 minutes, followed by 45 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, extension at 72°C for 45 seconds and a nal extension at 72°C for 10 minutes. The amplicons were assessed using 2% Agarose gel (SeaKem ® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure ® PCR product puri cation kit (Roche Molecular Diagnostics, Switzerland).
4
Characterization of EGFR Exons 18-21
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In a 20μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5μM). Approximately 50ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18] . Thermal cycler settings included an initial denaturation of 95°C for 15 minutes, followed by 45 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, extension at 72°C for 45 seconds and a nal extension at 72°C for 10 minutes. The amplicons were assessed using 2% Agarose gel (SeaKem ® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure ® PCR product puri cation kit (Roche Molecular Diagnostics, Switzerland).
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