Genomic DNA was extracted from the CF-CRC cells using the QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany 51183), and fluorimetric quantification was performed (Qubit, Invitrogen, CA, USA). Proximal 5’-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene (RefSeq NM_000492.4, NG_016465.4) were sequenced using the Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA), as previously described [31 –33 (link)] and a genetic analyzer (ABI PRISM 3130xl; Applied Biosystems, Foster City, CA, USA). Genotype analysis was completed using multiplex ligation-dependent probe amplification (SALSA MLPA probemix P091 CFTR, MRC Holland, Amsterdam, the Netherlands, EKI-FAM, P091-100).
Sanger cycle sequencing protocol
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sanger cycle sequencing protocol is a laboratory technique used for DNA sequencing. It employs the chain-termination method to determine the nucleotide sequence of a DNA molecule.
Automatically generated - may contain errors
4 protocols using sanger cycle sequencing protocol
1
CFTR Gene Sequencing and Genotyping
2
CFTR Gene Sequencing and Analysis
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Genomic DNA was extracted from both the peripheral blood leucocytes of enrolled individuals and derived undifferentiated CF-CRC-AESC, using the QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany) and quantified using fluorimetry (Qubit, Invitrogen, CA, USA). The proximal 5′-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene (RefSeq NM_000492.4, NG_016465.4) were sequenced by a Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA) as previously described [21 (link),22 (link),23 (link)], using a genetic analyzer (ABI PRISM 3130xl; ThermoFisher Scientific). Genetic analysis was completed through multiplex ligation-dependent probe amplification (SALSA MLPA probemix P091 CFTR, MRC Holland, Amsterdam, The Netherlands) to unveil macrodeletions/macroduplications. The genotype of derived undifferentiated CF-CRC-AESC was confirmed using the same methodology. Results of mutational analysis have already been published [5 (link),19 (link)].
3
CFTR Gene Sequencing and Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Genomic DNA was extracted from the CF-CRC cells using the QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany 51183), and fluorimetric quantification was performed (Qubit, Invitrogen, CA, USA). Proximal 5’-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene (RefSeq NM_000492.4, NG_016465.4) were sequenced using the Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA), as previously described [31 –33 (link)] and a genetic analyzer (ABI PRISM 3130xl; Applied Biosystems, Foster City, CA, USA). Genotype analysis was completed using multiplex ligation-dependent probe amplification (SALSA MLPA probemix P091 CFTR, MRC Holland, Amsterdam, the Netherlands, EKI-FAM, P091-100).
4
CFTR Gene Sequencing and Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from the CF-CRC cells using the QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany 51183), and fluorimetric quantification was performed (Qubit, Invitrogen, CA, USA). Proximal 5’-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene (RefSeq NM_000492.4, NG_016465.4) were sequenced using the Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA), as previously described [31 –33 (link)] and a genetic analyzer (ABI PRISM 3130xl; Applied Biosystems, Foster City, CA, USA). Genotype analysis was completed using multiplex ligation-dependent probe amplification (SALSA MLPA probemix P091 CFTR, MRC Holland, Amsterdam, the Netherlands, EKI-FAM, P091-100).
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