Genomic DNA of C. militaris was extracted from fresh mycelium by using a cetyltrimethy-lammonium bromide method [18 ]. The ITS4R and ITS5F primers were used to amplify rDNA region spanning the ITS1, ITS2, and 5.8S rRNA gene [19 ]. The reaction mixture for PCR was consisted of 10× Taq PCR buffer 2 µL, 1.6 µL dNTPs (2.5 mM stock), 1 µL primer 1 (10 pmol/µL), 1 µL primer 2 (10 pmol/µL), 0.1 µL Taq DNA polymerase (Takara, Tokyo, Japan), 1 µL dimethyl sulfoxide, and 50 ng/µL template. PCR was performed using Sure Cycler 8800 Thermal Cycler (Agilent Technologies, Santa Clara, CA, USA) under the following conditions: one cycle of 5 min at 96℃, followed by 30 cycles of 96℃ for 40 sec, 48℃ for 40 sec, and 72℃ for 40 sec, and finishing with extension at 72℃ for 10 min. The amplified PCR products was purified by PCR Purification kit (GeneAll Biotech, Seoul, Korea) and sequenced by 3730xl DNA analyzer (Macrogen, Seoul, Korea). Phylogenetic tree was created among 12 C. militaris strains including Cordyceps bassinia. All sequence were aligned by MUSCLE and curated by Gblocks. Phylogenetic tree was created by MEGA ver. 6.06 using the maximum liklihood. Bootstrap analysis was conducted with 2,000 replicates.
3730xl dna analyzer
Manufactured by Macrogen
Sourced in Netherlands
The 3730XL DNA analyzer is a sequencing instrument used for high-throughput DNA analysis. It employs capillary electrophoresis technology to separate and detect fluorescently-labeled DNA fragments. The instrument can generate DNA sequence data with high accuracy and speed.
Automatically generated - may contain errors
Lab products found in correlation
Fastap, by Thermo Fisher Scientific (2 mentions)
Seqman v 5, by DNASTAR (2 mentions)
Exo 1, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Editseq, by DNASTAR (2 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
Surecycler 8800 thermal cycler, by Agilent Technologies (1 mentions)
Pcr purification kit, by GeneAll (1 mentions)
Sequencher v5, by Gene Codes (1 mentions)
Hap 2, by Takara Bio (1 mentions)
Pcr purification kit, by Biofact (1 mentions)
Accupower pcr premix kit, by Bioneer (1 mentions)
6 protocols using 3730xl dna analyzer
1
Phylogenetic Analysis of Cordyceps militaris
2
Sanger Sequencing of HSPB1 Mutations
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Pathogenic mutations (404C>T and 545C>T) in HSPB1 gene from patients iPSCs were confirmed by Sanger sequencing using a 3730xl DNA Analyzer (Macrogen Inc., Seoul, Korea) and analyzed using Sequencher v.5.2.3 (GeneCodes Corporation, Ann Arbor, MI, USA). The primers used for amplifying and sequencing are as follows: 5′-TTT CTG AGC AGA CGT CCA GA-3′ (forward) and 5′-CTT TAC TTG GCG GCA GTC TC-3′ (reverse).
3
Microbial Diversity Analysis via T-RFLP
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The purified bacterial DNA from feces was amplified via polymerase chain reaction (PCR) using an AccuPower PCR PreMix kit (Bioneer, Seoul, South Korea) and broad-ranged primers: 27F-FAM (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Macrogen, Seoul, South Korea). The conditions for PCR amplification reactions were as follows: initial denaturation for 3 min at 94 °C; 30 cycles of amplification encompassing denaturation at 94 °C for 1 min, annealing at 53 °C for 45 s, and elongation at 72 °C for 2 min followed by a final extension at 72 °C for 10 min. The PCR products were purified using a PCR purification kit (BIOFACT, Seoul, South Korea) according to the kit manufacturer’s protocol, and the purified products were subjected to digestion with restriction enzyme Hap II (Takara, Kyoto, Japan). Finally, the digested DNA products were submitted to Macrogen Korea (Macrogen, Seoul, South Korea) for T-RFLP-based analysis of the microbial population using a 3730XL DNA analyzer (BigDye v3.1, Macrogen, Seoul, South Korea). The T-RFLP electropherograms were visualized using GeneScan 3.7 software (Thermo Fisher Scientific, Carlsbad, CA, USA) and analyzed using XLStat software (Addinsoft, Brooklyn, NY, USA).
4
Sanger Sequencing of PCR Amplicons
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PCR amplicons were visualized on a 2% agarose gel using Sybr green (Life Technologies Europe, Bleiswijk, the Netherlands) under UV light (Uvitec, Cambridge, United Kingdom). PCR products of the expected sizes were purified using 0.2 μL of FastAP (thermosensitive alkaline phosphatase) and 0.2 μL of Exo I (exonuclease I from Escherichia coli ) enzymes (Thermo Fisher Scientific, Waltham, MA, USA). Enzymatic digestion was carried out in a thermocycler at 37°C for 15 min, followed by enzyme inactivation at 80°C for 15 min. The purified PCR products were directly sequenced via the Sanger sequencing method by Macrogen, Inc. (Amsterdam, the Netherlands), on an automatic 3730XL DNA analyzer (https://www.macrogen-europe.com/services/sanger-sequencing/standard ). The obtained sequences were verified by the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and adjusted using Sequence Scanner v2.0 (https://products.appliedbiosystems.com ). The EditSeq and SeqMan v5.05 programs (DNASTAR Inc., Madison, WI, USA) were used to assemble the sequences.
5
Identifying Protein-Protein Interactions
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Using the specific primer Matchmaker 5′ AD, we sequenced the cDNA of pGADT7 prepared from mRNA containing information regarding PPIs with C4 protein in the pGBKT7 vector, using a 3730XL DNA analyzer (Macrogen, Seoul, Korea).
6
Sanger Sequencing of PCR Amplicons
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PCR amplicons were visualized on a 2% agarose gel using Sybr green (Life Technologies Europe, Bleiswijk, the Netherlands) under UV light (Uvitec, Cambridge, United Kingdom). PCR products of the expected sizes were purified using 0.2 μL of FastAP (thermosensitive alkaline phosphatase) and 0.2 μL of Exo I (exonuclease I from Escherichia coli ) enzymes (Thermo Fisher Scientific, Waltham, MA, USA). Enzymatic digestion was carried out in a thermocycler at 37°C for 15 min, followed by enzyme inactivation at 80°C for 15 min. The purified PCR products were directly sequenced via the Sanger sequencing method by Macrogen, Inc. (Amsterdam, the Netherlands), on an automatic 3730XL DNA analyzer (https://www.macrogen-europe.com/services/sanger-sequencing/standard ). The obtained sequences were verified by the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and adjusted using Sequence Scanner v2.0 (https://products.appliedbiosystems.com ). The EditSeq and SeqMan v5.05 programs (DNASTAR Inc., Madison, WI, USA) were used to assemble the sequences.
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