Astatotilapia burtoni fhl2a and fhl2b coding fragments were amplified by PCR (for primer information see Supplementary Table 3 ) using Phusion® Master Mix with HF Buffer (New England BioLabs, USA) following the manufacturer’s guidelines. These fragments were cloned into pCR4-TOPO TA vector using the TOPO® TA cloning kit (Invitrogen, USA). Plasmid extractions were done with GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, USA). RNA probes were synthetized with the DIG RNA labeling kit (SP6/T7) (Roche, Switzerland). The insertion and direction of the fragments was confirmed by Sanger sequencing using M13 primers (available with the cloning kit) and BigDye® terminator reaction chemistry (Applied Biosystems, USA) on an AB3130xl genetic analyzer (Applied Biosystems, USA). In situ hybridization was performed in 12 fins from A. burtoni males, six for fhl2a and six for fhl2b. The protocol was executed as described in ref. 16 (link), except for an intermediate proteinase K treatment (20 minutes at a final concentration of 15 μg/ml) and for the hybridization temperature (65°C).
Bigdye terminator reaction chemistry
Manufactured by Thermo Fisher Scientific
Sourced in United States
BigDye® terminator reaction chemistry is a DNA sequencing technology developed by Thermo Fisher Scientific. It is a core component used in various DNA sequencing applications. The product provides the necessary reagents and protocols for performing DNA sequencing reactions.
Automatically generated - may contain errors
Lab products found in correlation
Ab 3130xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
Genelute plasmid miniprep kit, by Merck Group (2 mentions)
Dig rna labeling kit sp6 t7, by Roche (1 mentions)
Phusion master mix with hf buffer, by New England Biolabs (1 mentions)
Dig rna labelling kit sp6 t7, by Roche (1 mentions)
2 protocols using bigdye terminator reaction chemistry
1
Astatotilapia burtoni fhl2a and fhl2b Expression
2
Cloning and in situ Hybridization of fhl2a and fhl2b in Astatotilapia burtoni
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A. burtoni fhl2a and fhl2b coding fragments were amplified by PCR (for primer information, see Supplementary Table 3 ) using Phusion Master Mix with High Fidelity buffer (New England BioLabs, USA) following the manufacturer’s guidelines. These fragments were cloned into pCR4-TOPO TA vector using the TOPO TA cloning kit (Invitrogen). Plasmid extractions were done with GenElute Plasmid Miniprep Kit (Sigma-Aldrich). RNA probes were synthetized with the DIG RNA labelling kit (SP6/T7) (Roche). The insertion and direction of the fragments was confirmed by Sanger sequencing using M13 primers (available with the cloning kit) and BigDye terminator reaction chemistry (Applied Biosystems) on an AB3130xl Genetic Analyzer (Applied Biosystems). In situ hybridization was performed in 12 fins from A. burtoni males, six for fhl2a and six for fhl2b. The protocol was executed as described in ref. 16 (link), except for an intermediate proteinase K treatment (20 min at a final concentration of 15 μg ml−1) and for the hybridization temperature (65 °C).
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