For the generation of point-mutated EGFR variants, the pCMV3-EGFRwt plasmid (Sino Biological, Eschborn, Germany) was used as template. Briefly, a high fidelity Q5 polymerase (New England Biolabs, Ipswich, MA) was used to amplify the whole plasmid with complementary primer pairs, carrying the desired mutation in the form of mismatches to the original plasmid. PCR conditions were: 94 °C for 2 min, 21 cycles of 94 °C (30 sec), 55 °C (1 min) and 68 °C (30 sec/kb). After three cycles, the oligonucleotides for the introduction of the respective point-mutations were added; primer sequences are listed in supplementary table 1c . Afterwards, the PCR mix was treated with DpnI endonuclease (New England Biolabs) to remove parental DNA. The successful introduction of point-mutations was validated by Sanger Sequencing.
Pcmv3 egfrwt plasmid
Manufactured by Sino Biological
Sourced in Germany
The PCMV3-EGFRwt plasmid is a DNA construct that contains the coding sequence for the epidermal growth factor receptor (EGFR) protein under the control of the cytomegalovirus (CMV) promoter. This plasmid can be used for the expression of the wild-type EGFR protein in various cell lines.
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Lab products found in correlation
2 protocols using pcmv3 egfrwt plasmid
1
Generation of EGFR Mutant Variants
2
Generation of EGFR Mutant Variants
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For the generation of point-mutated EGFR variants, the pCMV3-EGFRwt plasmid (Sino Biological, Eschborn, Germany) was used as template. Briefly, a high fidelity Q5 polymerase (New England Biolabs, Ipswich, MA) was used to amplify the whole plasmid with complementary primer pairs, carrying the desired mutation in the form of mismatches to the original plasmid. PCR conditions were: 94 °C for 2 min, 21 cycles of 94 °C (30 sec), 55 °C (1 min) and 68 °C (30 sec/kb). After three cycles, the oligonucleotides for the introduction of the respective point-mutations were added; primer sequences are listed in supplementary table 1c . Afterwards, the PCR mix was treated with DpnI endonuclease (New England Biolabs) to remove parental DNA. The successful introduction of point-mutations was validated by Sanger Sequencing.
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