Genomic DNA was extracted from tissue samples using a TIANamp FFPE DNA kit (Tiangen Biotech Co., Ltd.), in order to genotype the six most common HPV subtypes (HPV-16, −18, −31, −45, −52 and −58). PCR amplification was conducted using an E6 Nested Multiplex PCR to detect HPV in the tissue samples (29 (link)). The nested PCR utilized MY09-MY11 consensus primers, and the resultant PCR products served as a template for the nested PCRs using E6 genotype-specific primers to amplify HPV-16, −18, −31, −45, −52 and −58. Subsequently, the PCR products were analyzed using 2% agarose gel electrophoresis and stained with ethidium bromide (Tiangen Biotech Co., Ltd.), and, finally sequenced according to a previous study (29 (link)).
Ethidium bromide
Manufactured by Tiangen Biotech
Ethidium bromide is a fluorescent dye used in molecular biology laboratories to visualize and quantify nucleic acids, such as DNA and RNA, during gel electrophoresis. It binds to the nucleic acids and emits a distinctive orange-red fluorescence when exposed to ultraviolet light.
Automatically generated - may contain errors
2 protocols using ethidium bromide
1
HPV Genotyping from FFPE Tissue
2
DNA Oligonucleotide Synthesis and Purification
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All oligonucleotides used in
this experiment (seeTable S1 ) were synthesized
and purified from Sangon Biotech. Co. Ltd. (Shanghai, China). T4 DNA
ligase and Phi29 DNA polymerase were obtained from New England Biolabs
(NEB, Beijing, China). dNTPs, dGTP, dTTP, ethidium bromide, and 20
bp DNA ladder were obtained from Tiangen Biotech. Co. Ltd. (Beijing,
China). The other reagents in this experiment are shown in part 1
of theSupporting Information . Diethypyrocarbonate-treated
water (DNase, RNase free) obtained from Beyotime Institute of Biotechnology
(Shanghai, China) was used in all experiments.
this experiment (see
and purified from Sangon Biotech. Co. Ltd. (Shanghai, China). T4 DNA
ligase and Phi29 DNA polymerase were obtained from New England Biolabs
(NEB, Beijing, China). dNTPs, dGTP, dTTP, ethidium bromide, and 20
bp DNA ladder were obtained from Tiangen Biotech. Co. Ltd. (Beijing,
China). The other reagents in this experiment are shown in part 1
of the
water (DNase, RNase free) obtained from Beyotime Institute of Biotechnology
(Shanghai, China) was used in all experiments.
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