Total genomic DNA was isolated from 20 to 30 mg of the leaf samples or 50~100 mg of the medicinal materials using a Hi-DNAsecure Plant Kit (Tiangen Biotech Co., Beijing, China). The PCR amplification was performed in a 25 μL reaction mixture that contained 2 μL genomic DNA, 12.5 μL PCR MasterMix (Aidlab Biotechnologies Co., Beijing, China), 8.5 μL ddH2O, and 1 μL each of the forward and reverse primers (2.5 μM, synthesized by Sangon Co., China). The primers and the reaction conditions followed previously published methods for ITS2 and psbA-trnH (Chen et al., 2010 (link); Group et al., 2011 (link)). The PCR products were purified using the QIAquick PCR purification kit (Tiangen Biotech, Beijing, China), and the bidirectional sequencing was accomplished with an ABI 3730XL sequencer using the original amplification primer.
Pcr master mix
Manufactured by Aidlab
Sourced in China
The 2 × PCR Master Mix is a ready-to-use solution designed for the amplification of DNA sequences using the Polymerase Chain Reaction (PCR) technique. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, required for efficient PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Hi dnasecure plant kit, by Tiangen Biotech (1 mentions)
Qiaquick pcr purification kit, by Tiangen Biotech (1 mentions)
T vector, by Takara Bio (1 mentions)
3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
Primer 6, by Premier Biosoft (1 mentions)
Gelred, by Mei5 Biotechnology (1 mentions)
Plant genome dna kit, by Tiangen Biotech (1 mentions)
5 protocols using pcr master mix
1
Plant Genomic DNA Extraction and PCR Amplification
2
Amplifying A. convolvuli Mitogenome
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To amplify the A. convolvuli mitogenome, nine pairs of primers were designed from full-length mitogenome sequences of several lepidopteran species and then synthesized (Beijing Sunbiotech Co., Ltd.; Beijing, China) (Table 2). All PCRs were performed in 50 µL reaction volumes, which contained 25 µL PCR Master mix (2 ×; Aidlab Co.; Beijing, China), 1.5 µL extracted DNA as a template, 2 µL of each primer (10 µM), and 19.5 µL sterilized distilled water. The PCR was performed under the following conditions: an initial denaturation for 4 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 40 s at 46-58 °C (depending on the primer combination), 1-3 min (depending on the putative length of the fragments) at 72 °C, and a 10 min final extension at 72 °C.
The PCR products were separated by agarose gel electrophoresis (1% w/v) and were purified using a DNA gel extraction kit (TransGen Co.; Beijing, China) . The obtained fragments were cloned using T-vector (Takara Co.; Dalian, China) in XL-1 blue competent cells (TransGen Co.; Beijing, China) . The positive recombinant clone with an insert was sequenced at least three times (Invitrogen Co., Ltd.; Shanghai, D r a f t 5 China).
The PCR products were separated by agarose gel electrophoresis (1% w/v) and were purified using a DNA gel extraction kit (TransGen Co.; Beijing, China) . The obtained fragments were cloned using T-vector (Takara Co.; Dalian, China) in XL-1 blue competent cells (TransGen Co.; Beijing, China) . The positive recombinant clone with an insert was sequenced at least three times (Invitrogen Co., Ltd.; Shanghai, D r a f t 5 China).
3
Identification of S. chinensis by ITS2
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We randomly selected one sample (about 40 mg) in each batch of commercial crude drugs and 1–3 samples (200–300 mg) in each batch of Chinese patent medicines. The samples were ground into powder using a FastPrep bead mill (Retsch MM400). DNA was extracted from powder using the Plant Universal Genomic DNA Kit and amplified by ITS2F. (5′-ATGCGATACTTGGTGTGAAT-3′) /WWZ5R (5′-GCTCCTCGCAAACACCATAC-3′) primer pairs that were newly designed by Primer 6.0 software (Premier Biosoft International, Palo Alto, CA, USA) and specific for S. chinensis and its closely related species. The location of primer pairs is shown in Figure 2 . PCR was performed in a 25 µL reaction system containing 2 µL (about 30 ng) of DNA templates, 1.0 µL of primer ITS2F/WWZ5R (2.5 µmol/L), 12.5 µL of 2× PCR Master Mix (Aidlab Biotechnologies Co., Ltd., Beijing, China), and double-distilled water. The reactions were performed with the same thermal program that was used for the other materials. Purified PCR products were sequenced in both directions with the newly designed primer pairs by the Sanger sequencing method on a 3730 XL sequencer (Applied Biosystems, Inc., Foster City, CA, USA).
4
Molecular identification of Ephedra using PCR
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The primer pair MH-1F (5′-TCATCGAGTCTTTGAACGC-3′)/MH-1R (5′- ATGCGAAGGTCCCCTTTT-3′) was designed using Primer Premier 6.0 software (Premier Co., Palo Alto, CA, USA) for the amplification of the short DNA fragments (~150 bp) containing the nucleotide signature of Ephedra L. Polymerase chain reactions (PCR) were performed in a 25-µL system consisting of 12.5 µL of 2 × PCR Master Mix (Aidlab Biotechnologies Co., Beijing, China), 1.0 μL of forward/reverse primers (MH-1F/MH-1R, 2.5 μM), and 2.0 μL of DNA templates and filled with double-distilled water. The reactions were then performed as follows: 94 °C for 5 min; followed by 35 cycles of 94 °C for 45 s, 56 °C for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. The PCR products were examined via 1% (w/v) agarose gel electrophoresis that had been prestained by GelRed (Mei5 Biotechnology Co., Beijing, China) and bidirectionally sequenced using an ABI 3730XL sequencer (Applied Biosystems Co., Foster City, CA, USA) at the Major Engineering laboratory of Chinese Academy of Agricultural Sciences (Beijing, China).
5
DNA Extraction and Amplification of Herbal Materials
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The surface of all herbal materials was cleaned with 75% ethanol to avoid fungal DNA contamination. About 60 mg of the materials were cut into pieces, added with 10% polyvinylpyrrolidone (PVP), and then ground with a FastPrep bead mill (Retsch MM400, Germany). The total DNA was extracted with a Plant Genome DNA Kit (Tiangen Biotech Co., China) in accordance with the manufacturer's instructions. The ITS2 sequences were amplified using universal primers ITS-S2F (5′-ATGCGATACTTGGTGTGAAT-3′) and ITS-S3R (5′-GACGCTTCTCCAGACTACAAT-3′) as previously described (Chen et al., 2010 (link)). Polymerase chain reaction (PCR) amplification was performed in a 25 μL reaction mixture containing 12.5 μL of 2 × PCR Master Mix (Aidlab Biotechnologies Co., Ltd.), 1.0 μL of each primer (2.5 μM), 2 μL (about 30 ng) of DNA templates, and filled with double-distilled water. The reactions were performed with the following thermal program: 94°C for 5 min and 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, followed by 72°C for 10 min. The PCR products were sequenced by the Major Engineering laboratory of the Chinese Academy of Agricultural Sciences (Beijing, China).
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