Total RNAs were extracted from the periphery blood samples of the patients and unaffected control subjects, using the QIAamp RNA Blood Mini Kit (QIAGEN). cDNA were synthesised from 8 mg total RNA using GoScript Reverse Transcription System (Promega, Madison, USA) according to manufacturer’s protocol. RT-PCR primers were designed on Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/ ): RT-exon-9F (5′-GTGTCTGGTTTCTTGCCGTG-3′); RT-exon-11R (5′-GGCACTTCATGAACAC TCTCT-3′); RT-exon13F (5′-GCCCTTCATGCACAGTCATT-3′); RT-exon16R (5′-ACAGGAAAGGAGTCGAGAGG-3′). The 50 μL reaction system contained 40 ng cDNA, 10 pmol of each primer and 25 μL 2×Taq PCR Master Mix (TransGen Biotech, Beijing, China). DNA amplifications were performed with denaturing at 95°C for 5 min, followed by 33 cycles of a denaturing step at 95° C, an annealing step at 60°C and an extension step at 72°C, each for 30 s. A final extension step at 72°C was performed for 7 min. After purification, amplicons were sequenced using forward and reverse primers on an ABI 3730 Genetic Analyzer (ABI, Foster City, California, USA). Sequences were assembled and analysed using Lasergene SeqMan software (DNASTAR, Madison, Wisconsin, USA).
Taq pcr master mix
Manufactured by Transgene
Sourced in China
2×Taq PCR Master Mix is a ready-to-use solution for performing polymerase chain reaction (PCR) amplification. It contains Taq DNA polymerase, buffer, MgCl2, and dNTPs in a balanced concentration, optimized for efficient and reliable PCR.
Automatically generated - may contain errors
Lab products found in correlation
Goscript reverse transcription system, by Promega (1 mentions)
Qiaamp rna blood mini kit, by Qiagen (1 mentions)
Seqman software, by DNASTAR (1 mentions)
Transstart top green qpcr supermix kit, by Transgene (1 mentions)
Gusblue kit, by Huayueyang (1 mentions)
Genemapper, by Thermo Fisher Scientific (1 mentions)
Abi 3730 dna sequencer, by Thermo Fisher Scientific (1 mentions)
Easypure pcr purification kit, by Transgene (1 mentions)
Easypure bacteria genomic dna kit, by Transgene (1 mentions)
Barnstead labtower edi water purification system, by Thermo Fisher Scientific (1 mentions)
Hplc grade acetonitrile, by Dikma Technologies (1 mentions)
Applied biosystems 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
6 protocols using taq pcr master mix
1
RNA Extraction and RT-PCR Analysis
2
Overexpression of PscCYP716A1 in Poplar
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The acquired PscCYP716A1 sequence was inserted into the downstream position of the CaMV 35S promoter and transferred to the pCAMBIA2301 vector through the double enzyme (SacI and XbaI) digestion method described in the previous report (Wang et al., 2011 (link)). The resulting construct was introduced into the genome of 84 k poplars using the Agrobacterium-mediated leaf dish transformation method. The survival poplars were screened in WPM with 40 mg·L−1 kanamycin and 300 mg·L−1 cephalosporin for 15 days. After 6 months, we obtained 25 kanamycin-resistant poplars. To identify and acquire transgenic poplars, the PCR analysis was performed with 2 × Taq PCR Mastermix (KT201, Transgene, Beijing, China) and a pair of vector-specific primers, and the GUS staining was performed with the GUS blue kit (Huayueyang, Beijing, China). To make the expression level of PscCYP716A1 in different transgenic 84 k poplars clear, the RT-PCR analysis was performed with the TransStart® Top Green qPCR SuperMix Kit (AQ132-11, Transgene, Beijing, China). Finally, we obtained a total of nine overexpression lines named as POE-(1-9), two of which were selected as experimental materials for subsequent study.
3
Genotyping and Sequencing of Wild Soybean
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To reduce experimental expenses, genotyping was performed for 43 representive from 53 sampled wild soybean populations using 20 nSSRs, as in previous study (Table S1 , He et al.31 (link)). PCR reactions were performed in 15 μL of reaction containing 30–50 ng genomic DNA, 0.6 μM of each primer, 7.5 μL 2 × Taq PCR MasterMix (Transgen, Beijing, China). PCR amplifications were conducted under the following conditions: 94 °C for 2 min; 35 cycles at 94 °C for 30 s, 50 °C for 40 s, and 72 °C for 1 min; followed by a final extension step at 72 °C for 7 min. PCR products were separated on an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, California, USA). Fragment sizes were scored automatically using the program Genemapper (Applied Biosystems).
The plastid trnQ-rps16 was amplified from 599 individuals representing 52 of 53 populations (we failed to amplify this locus from population J5) using a primer pair of trnQ (GCGTGGCCAAGYGGTAAGGC) and rps16 (GTTGCTTTYTACCACATCGTTT)66 (link). TrnQ-rps16 was amplified and sequenced following the methods of Shaw, et al.66 (link). The PCR products were purified with an EasyPure PCR Purification Kit (TransGen). Purified PCR products were sequenced directly on an ABI 3730 sequencer.
The plastid trnQ-rps16 was amplified from 599 individuals representing 52 of 53 populations (we failed to amplify this locus from population J5) using a primer pair of trnQ (GCGTGGCCAAGYGGTAAGGC) and rps16 (GTTGCTTTYTACCACATCGTTT)66 (link). TrnQ-rps16 was amplified and sequenced following the methods of Shaw, et al.66 (link). The PCR products were purified with an EasyPure PCR Purification Kit (TransGen). Purified PCR products were sequenced directly on an ABI 3730 sequencer.
4
Bacterial Strain Identification and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The single most effective antagonistic bacterial strain (XH-9) showing a wide broadspectrum against phytopathogens was selected for further characterization, based on sequence analysis of 16S rDNA gene (11) . The bacterium was grown in Luria-Bertani(LB)broth culture at 28°C ± 2°C for 24 h and was then centrifuged at 3480 × g for 2 min. DNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. The 16S rDNA gene sequences from bacterial genomic DNA samples were amplified using universal primers 27F and 1492R (35) . PCR amplification was performed in 50-μL reactions containing 25 μL of 2× Taq PCR Master Mix (TransGen Biotech, Beijing, China), 2 μL of each primer (10 μM), 2 μL of template DNA (10 ng/µL), and 19 μL of ddH 2 O. PCR amplification was performed under the following conditions: initial denaturation at 94°C for 3 min; 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min. The obtained amplicons were sequenced by a commercial sequencing company (Sangon Biotech, Shanghai, China). The obtained gene sequences were analyzed and BLAST searched againstwith the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and a phylogenetic tree was constructed using MEGA 5.1 software (36) .
5
Quantitative Analysis of Fumonisins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Taq PCR Master Mix, DNA Marker DL2000 were acquired from TransGen Biotech (Beijing, China). Sodium hypochlorite solution, dextrose, agar were purchased from BBI Life Sciences Corporation. FB standards (FB1 and FB2) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol used for sample preparation and mobile phase were supplied by Dikma (Lake Forest, CA, USA). 2-Mercaptoethanol, OPA, and potassium borate buffer o-phthalaldehyde diluent (OD104) were purchased from Pickering Laboratories (Mountain View, CA, USA). Ultrapure water was prepared in all analytical steps using a Barnstead LabTower EDI water purification system (ThermoFisher Scientific, Waltham, MA, USA).
6
RNA Extraction and qRT-PCR Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The detailed steps of RNA extraction and qRT-PCR were described in our previous study (18 (link)). TRIzol reagent was purchased from Invitrogen. Applied Biosystems 7500 Fast Real-Time PCR System and SYBR-Green PCR Master Mix were purchased from Applied Biosystems (Foster City, CA, USA), Taq PCR Master Mix was purchased from TransGen (Beijing, China). 7900HT Fast Real-Time PCR System was obtained from Applied Biosystems.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!