To confirm the homologous integration of the deletion construct into the genome, gel-purified gDNA from potential Δpks1 and Δku80_Δpks1 transformants and their parental strains were digested overnight by PstI (Thermo). gDNA of Δku80 strains was digested overnight with AatII (Thermo). Digested gDNA was loaded on a 0.8% agarose gel, separated by electrophoresis, and transferred to a nylon membrane Hybond N+ (GE Healthcare). The probes for the upstream regions of pks1 (oligonucleotides 9 and 10) and ku80 (oligonucleotides 24 and 25) deletion constructs were synthetized using PCR DIG DNA Labelling Mix (Roche) and Taq DNA Polymerase (NEB). Probes were hybridized overnight in DIG Easy Hyb (Roche) at 42°C and detection was performed by chemiluminescence using anti-Digoxigenin-AP Fab fragments (Roche) and CDP-Star chemiluminescent substrate (Roche) in a ChemiDoc MP (Bio-Rad).
Pcr dig dna labelling mix
Manufactured by Roche
The PCR DIG DNA Labelling Mix is a ready-to-use solution for the incorporation of digoxigenin-labeled nucleotides into DNA during PCR amplification. It allows for the detection of specific DNA sequences in various applications such as Southern blotting, in situ hybridization, and colony/plaque screening.
Automatically generated - may contain errors
Lab products found in correlation
Cdp star chemiluminescent substrate, by Roche (2 mentions)
Taq dna polymerase, by New England Biolabs (2 mentions)
Dig easy hyb, by Roche (2 mentions)
Anti digoxigenin ap fab fragment, by Roche (2 mentions)
Aatii, by Thermo Fisher Scientific (2 mentions)
Hybond n, by GE Healthcare (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
2 protocols using pcr dig dna labelling mix
1
Verifying Homologous Gene Deletion in Fungi
2
Verifying Homologous Gene Deletion in Fungi
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To confirm the homologous integration of the deletion construct into the genome, gel-purified gDNA from potential Δpks1 and Δku80_Δpks1 transformants and their parental strains were digested overnight by PstI (Thermo). gDNA of Δku80 strains was digested overnight with AatII (Thermo). Digested gDNA was loaded on a 0.8% agarose gel, separated by electrophoresis, and transferred to a nylon membrane Hybond N+ (GE Healthcare). The probes for the upstream regions of pks1 (oligonucleotides 9 and 10) and ku80 (oligonucleotides 24 and 25) deletion constructs were synthetized using PCR DIG DNA Labelling Mix (Roche) and Taq DNA Polymerase (NEB). Probes were hybridized overnight in DIG Easy Hyb (Roche) at 42°C and detection was performed by chemiluminescence using anti-Digoxigenin-AP Fab fragments (Roche) and CDP-Star chemiluminescent substrate (Roche) in a ChemiDoc MP (Bio-Rad).
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