1 μg of total RNA was reverse-transcribed using QuantiMir RT Kit (System Biosciences). The cDNA was diluted (100-fold) and amplified using PCR Supermix (Invitrogen) or iTaq SYBR Green Supermix with Rox (Bio-Rad) on GeneAmp PCR System 9700 or on ABI 7300 Real Time PCR system.
Geneamp pcr system 9700
Manufactured by Bio-Rad
Sourced in United States
The GeneAmp PCR System 9700 is a thermal cycler designed for performing polymerase chain reaction (PCR) experiments. It is capable of precisely controlling temperature profiles to facilitate the amplification of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Cfx96 real time pcr system, by Bio-Rad (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Quantimir rt kit, by System Biosciences (1 mentions)
Pcr supermix, by Thermo Fisher Scientific (1 mentions)
Itaq sybr green supermix with rox, by Bio-Rad (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rnafast200 kit, by Fastagen (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
Cfx connect real time pcr, by Bio-Rad (1 mentions)
Genemarker software, by SoftGenetics (1 mentions)
Sybr green mix, by Bio-Rad (1 mentions)
7 protocols using geneamp pcr system 9700
1
Reverse Transcription and qPCR Analysis
2
Quantitative Analysis of mGluR1 and mGluR5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of the hippocampal tissues were extracted by the RNA fast 200 kit (Fastagen, Shanghai, China). Then, reverse transcription was conducted where 1000 nanograms of RNA from each sample were reverse transcribed. Moreover, the reverse transcription reaction was performed utilizing the Revert Aid First Strand cDNA Synthesis Kit (Thermo, Waltham, MA, USA). The reaction was performed briefly in the GeneAmp PCR System 9700 (Bio-Rad, Hercules, CA, USA). The quantitative real-time PCR was performed in the CFX Connect Real-Time PCR (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR assays were conducted using Thunderbird TM SYBR® qPCR Mix (Toyobo, Osaka, Japan). The specific gene primers were then synthesized by the Sangon Biological Engineering Co., Ltd. (Shanghai, China). Moreover, the production lengths of primer sequences utilized in this experiment are reflected in Table 1 . The relative expressions of mGluR1 and mGluR5 were thus calculated through the adaptation of 2−∆ΔCt methods.
3
Chicken INHA Gene Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers for amplifying and sequencing the chicken INHA gene (Table 1 ) were designed with Primer Premier 5.0 software based on the complete DNA sequences of Gallus gallus INHA genes (ENSGALG00000054770). A DNA pool containing 100 ng DNA from each of the 60 least closely related chickens was constructed. PCR was carried out using a Gene Amp PCR System 9700 (Bio-Rad, Hercules, CA, USA) thermal cycler in a final volume of 25 μL containing 8.6 μL distilled H2O, 15 μL 2×Taq PCR Master Mix (including Mg2+, dNTPs, and Taq DNA polymerase; Beijing Tian Wei Biology Technique Corporation, Beijing, China), 0.3 μL each primer (10 nmol/L), and 0.8 μL chicken genomic DNA template. PCR was performed with the following cycling conditions: denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 56-60°Cfor 35 s (Table 1 ), and 72°C for 40 s, followed by a final extension at 72°C for 5 min. PCR products were purified and sequenced via an ABI 377 DNA sequencer (Shanghai Sangon Biological Engineering Technology, Shanghai, China). All sequences were edited, assembled, and aligned with DNASTAR and Codon Code Aligner software (http://www.codoncode.com/aligner ). SNPs were identified by the presence of multiple peaks at the same base by direct sequencing.
4
Optimized PCR Protocol for CRBP1 and CRBP3 Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primers were designed on the basis of DNA sequence of the CRBP1 (Accession no. NC_006096.2) and the CRBP3 (Accession no. NC_006088.2) using the oligo nucleotide design tool Primer 5.0 software (Table 1 ). Primer synthesis was completed by Shanghai Yingjun Biotechnology Co. Ltd. (Shanghai, China). PCR reactions were performed using the Gene Amp PCR System 9700 (Bio-Rad, Hercules, CA, USA) thermal cycler in a final volume of 10 μL reaction containing 10 to 100 ng genomic DNA, 5 μL 2×Taq PCR MasterMix (including Mg2+, dNTPs, Taq DNA polymerase; Beijing TIAN WEI Biology Technique Corporation, Beijing, China), 3.4 μL ddH2O and 0.4 μL of each primer (10 pmol/μL). The cycling protocol was 4 min at 94°C, 35 cycles of denaturing at 94°C for 30 s, annealing at X°C (Table 1 ) for 30 s, extending at 72°C for 1 min, with a final extension at 72°C for 8 min. The PCR products were characterized in 1% agarose gel electrophoresis.
5
Transferability and Polymorphism of Populus SSR Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 920 SSR primers were downloaded from the Populus Molecular Genetics Cooperative (http://www.ornl.gov; GCPM and PMGC primers) and the Oak Ridge National Laboratory (ORPM primers; Tuskan et al., 2004) (link). DNA extracted from two individuals was amplified to test the transferability and suitability of SSRs in P. talassica. Eight individuals were then used to test for polymorphisms in markers that exhibited robust amplification.
The forward primer of each pair was tagged with the universal M13 sequence (5'-TGTAAAACGACGGCCAGT-3') during synthesis. Each PCR used a 20-µL total volume containing 10 µL 2X Tap PCR Mix (Biomedtech, Beijing, China), 0.4 µL fluorescent-dyelabeled (FAM, HEX, TAMRA, ROX) M13 primer, 0.08 µL forward primer, 0.32 µL reverse primer, 7.2 µL ddH 2 O, and 2 µL (~20 ng) genomic DNA. PCR amplifications were performed in a GeneAmp ® PCR System 9700 thermal cycler (Bio-Rad Laboratories Inc., Beijing, China) using the following program: 94°C for 10 min; followed by 30 cycles of 30 s at 94°C, 40 s at 53°C, and 40 s at 72°C; then 10 cycles of 30 s at 94°C, 40 s at 50°C, and 40 s at 72°C; and a final extension at 72°C for 10 min. The PCR products were resolved using an ABI 3730XL DNA Analyzer by Genewiz Biotechnology Co., Ltd., (Beijing, China), and the data were analyzed using the Gene-Marker software (SoftGenetics LLC, State College, PA, USA).
The forward primer of each pair was tagged with the universal M13 sequence (5'-TGTAAAACGACGGCCAGT-3') during synthesis. Each PCR used a 20-µL total volume containing 10 µL 2X Tap PCR Mix (Biomedtech, Beijing, China), 0.4 µL fluorescent-dyelabeled (FAM, HEX, TAMRA, ROX) M13 primer, 0.08 µL forward primer, 0.32 µL reverse primer, 7.2 µL ddH 2 O, and 2 µL (~20 ng) genomic DNA. PCR amplifications were performed in a GeneAmp ® PCR System 9700 thermal cycler (Bio-Rad Laboratories Inc., Beijing, China) using the following program: 94°C for 10 min; followed by 30 cycles of 30 s at 94°C, 40 s at 53°C, and 40 s at 72°C; then 10 cycles of 30 s at 94°C, 40 s at 50°C, and 40 s at 72°C; and a final extension at 72°C for 10 min. The PCR products were resolved using an ABI 3730XL DNA Analyzer by Genewiz Biotechnology Co., Ltd., (Beijing, China), and the data were analyzed using the Gene-Marker software (SoftGenetics LLC, State College, PA, USA).
6
Pluripotency Gene Expression Analysis of Encapsulated mESCs
7
Quantifying Gene Expression in MCF-7 μtumors
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!