Rooted cuttings were grown in soil inside chambers with a 16 h photoperiod at 22 °C for 3 weeks. Leaf tissues were harvested in liquid nitrogen and ground using a motor and pestle. DNA extraction and purification were carried out using the Mini Genomic DNA Kit (IBI Scientific, Peosta, Iowa, USA). DNA was quantified using the Epoch spectrophotometer (Biotek, Winooski, VT) Powerwave XS2 with Gen 5.0 software. StBiP1, StBiP2, and StBiP3 genomic sequences were PCR amplified using gene-specific primers (Supplementary Table S4 online). Platinum SuperFi Green DNA Polymerase (Invitrogen) mix was used with PCR amplification conditions as follows; Initial denaturation 98 °C for 2 min, 98 °C 10 s, 60.9 °C 10 s, 72 °C for 2 min for 35 cycles, and final extension of 72 °C for 5 min. Amplified products were visualized using agarose gel (1%) electrophoresis. Sequencing was carried out using ABI 3,130 Genetic Analyzer (Applied Biosystems) using sequencing primers (Supplementary Table S4 online). The sequencing data was analyzed using Geneious Prime v. 2019.2.1. Gene sequences were deposited at NCBI Genbank under following accession numbers; MN982518 (StBiP1), MN982519 (StBiP2) and MN982520 (StBiP3).
Platinum superfi green dna polymerase
Manufactured by Thermo Fisher Scientific
Platinum SuperFi Green DNA polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It features a 3'→5' exonuclease proofreading activity for enhanced accuracy. This enzyme is supplied with a green fluorescent dye that enables real-time monitoring of amplification during PCR.
Automatically generated - may contain errors
6 protocols using platinum superfi green dna polymerase
1
Isolation and Sequencing of StBiP Genes
2
RT-PCR Profiling of A-to-I Editing
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Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to test for the presence of A-to-I editing in AZIN1, BLCAP, GLI1, SON and HTR2C mRNAs. To amplify AZIN1 and BLCAP sequences from bowhead and pig, we designed primers, which could be used for both species. Primer sequences for BMAZIN-EDF, BMAZIN-EDR, BMBLCAP-EDF and BMBLCAP-EDR and amplicon sizes are listed in S1 Table . For GLI1, primers BMGLI1-EDF and BMGLI1-EDR were used; for SON, the primers SSSON-AIF and SSSON-AIR were used; and for HTR2C, we employed the primers BMHTR2C-AIF and HTR2C-AIR (S1 Table ). PCR was performed with Invitrogen Platinum SuperFi Green DNA polymerase and 0.5μM primers. We used the following PCR program for BLCAP: 98°C for 30 secs followed by 35 cycles of 98°C for 10 secs and 72°C for 15 secs, finalizing with 72°C for 15 secs.
For AZIN PCR we used a three-step protocol consisting of 98°C for 30 secs followed by 35 cycles of 98°C for 10 secs, annealing at 65°C for 20 secs and 72°C for 15 secs, and finalizing with 72°C for 15 secs.
For AZIN PCR we used a three-step protocol consisting of 98°C for 30 secs followed by 35 cycles of 98°C for 10 secs, annealing at 65°C for 20 secs and 72°C for 15 secs, and finalizing with 72°C for 15 secs.
3
Amplification of Putative Editing Sites
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The cDNA fragments containing the putative editing sites of COG3, GRIA2, FLNA and FLNB were amplified using the primers SSCOG3-AIF and SSCOG3-AIR, BMCOG3-AIF and BMCOG3-AIR, SSGRIA2-AIF and SSGRIA-AIR, BMFLNA-AIF and BMFLNA-AIR and BMFLNB-AIF and BMFLNB-AIR, and BMIGFBP7-AIF and BMIGFBP7-AIR. Primers SAGRIA2-F and SAGRIA2-R were used to amplify the shark GRIA2 sequences. See S1 Table for sequence information. PCR was performed with Invitrogen Platinum SuperFi Green DNA polymerase and 0.5-μM primers. We used the following PCR program to amplify porcine and bowhead COG3 and FLNA amplicons: a three-step protocol consisting of 98°C for 30 secs followed by 35 cycles of 98°C for 10 secs, annealing at 65–72°C for 20 secs and 72°C for 15 secs, and finalizing with 72°C for 15 secs. Annealing temperatures were 66°C for COG3, 54°C for GRIA2, and 72°C for FLNA and FLNB, respectively.
4
Amplification of Porcine, Bowhead, and Shark NEIL1 Genes
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NEIL1-specific oligonucleotide primers derived from DNA sequences in exon 6 of the porcine and bowhead NEIL1 genes were used in a polymerase chain reaction (PCR). The sequences of the primers SSNEIL1-AIF, SSNEIL1-AIR, BMNEIL1-EF1, BMNEIL1-ER1, SANEIL1-AIF and SANEIL1-AIR are listed in S1 Table . PCR was performed with Invitrogen Platinum SuperFi Green DNA polymerase and 0.5-μM primers. We used the following PCR program to amplify porcine, bowhead and shark amplicons of 318 bp, 238 bp and 322 bp, respectively: 98°C for 30 secs followed by 32 cycles of 98°C for 10 secs and 72°C for 15 secs, finalizing with 72°C for 15 secs.
5
Porcine Huntingtin Protein UTR Analysis
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We used three oligonucleotide primer sets specific to huntingtin protein (HTT) to examine five edited adenosines located within the porcine 3’ untranslated region (UTR) sequence: HTT-F1 and HTT-R1, HTT-F3 and HTT-R3, and HTT10711-F and HTT10711-R (S1 Table ). PCR was performed with Invitrogen Platinum SuperFi Green DNA polymerase and 0.5-μM primers. We used the following PCR program to amplify the porcine HTT amplicons of 300 bp, 338 bp and 1054 bp: 98°C for 30 secs followed by 32 cycles of 98°C for 10 secs and 72°C for 15 secs, finalizing with 72°C for 15 secs.
6
Isolation and Sequencing of Potato BiP Genes
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DNA was extracted using rooted cuttings grown in soil inside chambers with a 16 h photoperiod at 22°C for three weeks. Briefly, tissues were harvested in liquid nitrogen and ground using a motor and pestle. DNA extraction and purification were carried out using Mini Genomic DNA Kit (IBI Scientific, Peosta, Iowa, USA). DNA quantification was carried out using the Epoch spectrophotometer (Biotek, Winooski, VT) Powerwave XS2 with Gen 5.0 software. PCR amplification of StBiP1, StBiP2, and StBiP3 was carried out using gene-specific primers (Table S4). Platinum™ SuperFi Green DNA Polymerase (Invitrogen) mix was used with PCR amplification conditions as follows; Initial denaturation 98 C for 2 min, 98 C 10 sec, 60.9 C 10 sec, 72 C for 2 min for 35 cycles, and final extension of 72 C for 5 mins. Amplified products were visualized using agarose gel (1%) electrophoresis. Sequencing was carried out using ABI 3130 Genetic Analyzer (Applied Biosystems) using sequencing primers (Table S4).
The sequencing data of each StBiP was analyzed using Geneious Prime® v. 2019.2.1. Gene sequences were deposited at NCBI Genbank under following accession numbers; MN982518 (StBiP1), MN982519 (StBiP2) and MN982520 (StBiP3)
The sequencing data of each StBiP was analyzed using Geneious Prime® v. 2019.2.1. Gene sequences were deposited at NCBI Genbank under following accession numbers; MN982518 (StBiP1), MN982519 (StBiP2) and MN982520 (StBiP3)
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