The human ASIC1a sequence was subcloned into a pSP65-derived vector containing 5′ and 3′ non-translated sequences of β globin to improve the stability and the expression in Xenopus laevis oocytes. Amino acid substitutions were generated by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase (KAPA Biosystems), using the Quikchange approach. Mutations were verified by sequencing (Microsynth). In vitro transcription was performed using the mMESSAGE mMACHINE SP6 kit (Ambion/Life Technologies).
Kapa hifi hotstart pcr polymerase
Manufactured by Roche
Sourced in Switzerland
KAPA HiFi HotStart PCR polymerase is a high-fidelity DNA polymerase enzyme designed for PCR amplification. It is capable of accurate DNA synthesis with enhanced proofreading activity.
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6 protocols using kapa hifi hotstart pcr polymerase
1
ASIC1a Expression in Xenopus Oocytes
2
Heterologous Expression of ASIC1a in Oocytes and Cells
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For the expression in Xenopus oocytes, the hASIC1a sequence (García-Añoveros et al., 1997 (link)) was sub-cloned into a pSP65-derived vector containing 5′ and 3′ non-translated sequences of β globin to improve the stability and the expression in Xenopus laevis oocytes. For the expression in chinese hamster ovary (CHO) cells, the human and mouse ASIC1a sequences were sub-cloned into the peak8 vector (Edge Biosystems, Gaithersburg, MD, USA). Amino acid substitutions were generated by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase (KAPA Biosystems), using the Quikchange approach. Mutations were verified by sequencing (Synergen Biotech). In vitro transcription was performed using the mMESSAGE mMACHINE SP6 kit (Ambion/Life Technologies).
3
Amplifying and Sequencing Genomic DNA
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For target site amplification, 100 ng of genomic DNA was amplified with KAPA HiFi HotStart PCR Polymerase (Roche, Basel, Switzerland). For deep sequencing library generation, amplicons were amplified a second time using TruSeq HT Dual Index Primers (Illumina, San Diego, CA, USA). Paired-end sequencing was performed using the Illumina Miniseq System. Indel frequencies were calculated at https://github.com/ibs-cge/maund .
4
ASIC1a Mutant Expression in Xenopus
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The human ASIC1a cDNA construct (García-Añoveros et al., 1997 (link)) was cloned into a pSP65 vector containing 5’- and 3’- untranslated sequences for expression in Xenopous oocytes. As reported recently, this construct contains the mutation G212D (Vaithia et al., 2019 (link)). Point mutations were introduced by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase (Roche Diagnostics, Rotkreuz, Switzerland). All mutations were verified by sequencing (Synergen Biotech). Complementary RNAs were synthetized in vitro using the mMESSAGE mMACHINE SP6 kit (Thermofisher).
5
Nested PCR for NGS Library Preparation
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Nested PCR was used to produce libraries for next-generation sequencing (NGS). The region of interest was first amplified by PCR using KAPA HiFi HotStart PCR polymerase (Roche). To generate NGS libraries, amplicons were amplified again using TruSeq DNA-RNA CD index-containing primers to label each fragment with adapter and index sequences. Final PCR products were purified using a PCR purification kit (MGmed) and sequenced using a MiniSeq sequencer (Illumina) with a GenerateFASTQ workflow. Primer sequences for targeted deep sequencing are listed in Supplementary Table 10 . Substitution and indel frequencies from targeted deep sequencing data were calculated with source code (https://github.com/ibs-cge/maund , written by BotBot Inc.).
6
Mutagenesis of ASIC1a cDNA for Xenopus expression
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The human ASIC1a cDNA construct (Garcia-Anoveros et al., 1997) was cloned into a pSP65
vector containing 5'-and 3'untranslated sequences for expression in Xenopous oocytes. As reported recently, this construct contains the mutation G212D (Vaithia et al., 2019) . Point mutations were introduced by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase (Roche Diagnostics, Rotkreuz, Switzerland). All mutations were verified by sequencing (Synergen Biotech). Complementary RNAs were synthetized in vitro using the mMESSAGE mMACHINE SP6 kit (Thermofisher).
vector containing 5'-and 3'untranslated sequences for expression in Xenopous oocytes. As reported recently, this construct contains the mutation G212D (Vaithia et al., 2019) . Point mutations were introduced by site-directed mutagenesis using KAPA HiFi HotStart PCR polymerase (Roche Diagnostics, Rotkreuz, Switzerland). All mutations were verified by sequencing (Synergen Biotech). Complementary RNAs were synthetized in vitro using the mMESSAGE mMACHINE SP6 kit (Thermofisher).
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