PCR amplification was done in 20 μL reaction mixture, comprising 10 μL of 2X Prime Taq premix (Genet Bio, Daejeon, Korea), 1.0 μL of each forward and reverse primers (10 pmoles), 2 μL of 100 ng genomic DNA as template and 6 μL of deionized distilled water. Primers are listed in Table 2 and Table S1 . The PCR conditions were as follows: 95 °C for 5 min, followed by amplifications of 30–35 cycles at 95 °C for 30 s, 58 and 60 °C for 30 s (specific Tm to respective primer sets in Table 2 and Table S1 ), 72 °C for 30 s and 72 °C for 5 min. Then, amplified PCR products were assessed by gel electrophoresis (1.5% agarose gel).
Prime taq premix
Manufactured by GeNet Bio
Prime Taq Premix is a ready-to-use, pre-mixed solution containing Taq DNA polymerase, buffer, dNTPs, and Mg2+ ions, designed for efficient and reliable DNA amplification in PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Ecodye, by Biofact (5 mentions)
Reverse transcription kit, by Elpis Biotech (3 mentions)
Qiazol lysis reagent, by Qiagen (3 mentions)
Superscript cdna premix kit 2, by GeNet Bio (2 mentions)
Suprimescript rt premix, by GeNet Bio (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Fusioncapt advance, by Vilber (2 mentions)
Fusion capt advance software, by Vilber (2 mentions)
Rotor gene q 2plex system, by Qiagen (2 mentions)
Reverse transcriptase premix, by Elpis Biotech (2 mentions)
Sybr green master mix, by Qiagen (2 mentions)
Sybr green, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Agarose gel, by Promega (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Hinfi restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Ibright cl1000 imaging system, by Thermo Fisher Scientific (1 mentions)
39 protocols using prime taq premix
1
PCR Amplification of Genomic DNA
2
Amplification of Biosynthetic Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three sets of degenerate primers were used for the amplification of genes encoding polyketide synthases I and II (PKS-I and PKS-II) and nonribosomal peptide synthetases (NRPS) (Table 1 ). The PCR reaction mixture that consisted of 20–200 ng bacteria genomic DNA, 10.0 μL of 2X Prime Taq Premix (Genet Bio, Korea), 10 pmoles of different primer sets (Table 1 ), and sterile ultrapure water was added to final volume of 20 μL. The PCR was performed using the Kyratec PCR Supercycler (Kyratec, Australia) with the following cycling conditions: (i) 94°C for 5 min; (ii) 30 cycles of 94°C for 1 min, 57°C (for K1F-M6R and KSα-KSβ) or 62°C (for A3F-A7R) for 1 min, and 72°C for 2 min; and (iii) 72°C for 5 min. The PCR amplification products were resolved using electrophoresis in 1.5% agarose gel (Promega, USA) and stained with ethidium bromide (0.5 μg mL−1) and viewed using Molecular Imager Chemidoc XRS System (Biorad, USA).
3
KRAS SNP Identification via PCR-RFLP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method for identification of KRAS SNPs. For KRAS rs61764370, the forward and reverse primers were 5`-GTGTCAGAGTCTCGCTCTTGTC-3` and 5`-AGACCACACTAGCACTACCTAAGGA-3`, respectively. For KRAS rs712 the primers were 5`-AAGGCATACTAGTACAAGTGGTAA-3` and 5`-TGTGTTCCCTCAATGTTTCAGT-3`.
In each 0.20 ml PCR reaction tube, we mixed 1 μl of genomic DNA (~100 ng/ml), 1 μl of each primer and 10 μl of 2X Prime Taq Premix (Genet Bio, Korea) in addition to 7 μl ddH2O. The PCR conditions were standardized as follows: 5 min preheating at 95°C, 30 cycles of 95°C for 30s, 62°C for rs712, and 58°C for rs61764370 for 30s, and 72 °C for 30s followed by a final extension step for 10 min at 72 °C. For rs61764370, the PCR product (10 μl) was digested using HinfI restriction enzyme (Thermo Scientific, Vilnius, Lithuania). The T allele was digested and produced 80, 135, 161 bp fragments, but the G allele produces 296 and 80-bp amplicons (Figure 1 ).
For KRAS rs712, 10 μl of PCR product was digested using TaqI (Thermo Scientific, Vilnius, Lithuania) restriction enzyme. Product size was 426-bp for T allele (undigested); G allele digested and produces 154-bp and 272-bp (Figure 2 ). To verify the genotyping quality, we randomly re-genotyped 20% of the samples and the genotypic were 100% concordant.
In each 0.20 ml PCR reaction tube, we mixed 1 μl of genomic DNA (~100 ng/ml), 1 μl of each primer and 10 μl of 2X Prime Taq Premix (Genet Bio, Korea) in addition to 7 μl ddH2O. The PCR conditions were standardized as follows: 5 min preheating at 95°C, 30 cycles of 95°C for 30s, 62°C for rs712, and 58°C for rs61764370 for 30s, and 72 °C for 30s followed by a final extension step for 10 min at 72 °C. For rs61764370, the PCR product (10 μl) was digested using HinfI restriction enzyme (Thermo Scientific, Vilnius, Lithuania). The T allele was digested and produced 80, 135, 161 bp fragments, but the G allele produces 296 and 80-bp amplicons (
For KRAS rs712, 10 μl of PCR product was digested using TaqI (Thermo Scientific, Vilnius, Lithuania) restriction enzyme. Product size was 426-bp for T allele (undigested); G allele digested and produces 154-bp and 272-bp (
4
Detection of TNF-α Polymorphisms by PCR-RFLP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TNF-α polymorphisms (-238 A/G, -308 A/G, -857 C/T, and -863 A/C) were detected by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). The primers were designed according to the data achieved from the NCBI data bank (http:www.ncbi.nlm.nih.gov) for identification of SNPs and listed in Table 1 . The restriction enzymes and the fragment length after digestion are shown in Table 1 . Every reaction contained 1 μL of each primer, 100 ng of template DNA, and 10 μL of 2X Prime Taq Premix (Genet Bio, Korea) and 7 μL of ddH2O in a 20 μL of total reaction volume.
The PCR conditions were as follows: initial denaturation at 95°C for 5 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58.5°C for -238 A/G, 59°C for -308 A/G, 63°C for -857 C/T, and 52°C for -863 A/C polymorphisms of the TNF-α for 30 seconds and extension at 72°C for 30 seconds, followed by a final extension step at 72°C for 5 minutes. Finally, the PCR products digested by the restriction enzymes (Fermentas, Vilnius, and Lithuania) and digested products were resolved by electrophoresis in 4 % agarose gel and stained with ethidium bromide (Figure 1A , B, C, and D).
The PCR conditions were as follows: initial denaturation at 95°C for 5 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58.5°C for -238 A/G, 59°C for -308 A/G, 63°C for -857 C/T, and 52°C for -863 A/C polymorphisms of the TNF-α for 30 seconds and extension at 72°C for 30 seconds, followed by a final extension step at 72°C for 5 minutes. Finally, the PCR products digested by the restriction enzymes (Fermentas, Vilnius, and Lithuania) and digested products were resolved by electrophoresis in 4 % agarose gel and stained with ethidium bromide (
5
Genotyping using PCR-RFLP Technique
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genotyping of the variants was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The primers sequences are listed in Table 2(Tab. 2) .
1 μl genomic DNA (~100 ng/μl), 1 μl (10 μM) forward and reverse primers, 10 μl 2X Prime Taq Premix (Genet Bio, Korea), and 7 μl ddH2O were added into a 0.20 ml PCR reaction tube. The PCR conditions were, initial denaturing step at 95 °C for 5 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing at appropriate temperature (Table 1(Tab. 1) ) for 30 s, extension at 72 °C for 30 s, and then a final extension step for 10 min at 72 °C. Then, 10 μl of PCR product was digested by suitable restriction enzyme (Table 1(Tab. 1) ) based on the manufacturer's procedure (Figure 1(Fig. 1) ). The digested products were electrophoresed on agarose gel containing 0.5 μg/ mL ethidium bromide, visualized on a UV transilluminator and photograph was taken (Figure 1(Fig. 1) ). For the quality control of genotyping; approximately, 20 % of the random samples were blindly regenotyped and the reproducibility was 100 %.
1 μl genomic DNA (~100 ng/μl), 1 μl (10 μM) forward and reverse primers, 10 μl 2X Prime Taq Premix (Genet Bio, Korea), and 7 μl ddH2O were added into a 0.20 ml PCR reaction tube. The PCR conditions were, initial denaturing step at 95 °C for 5 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing at appropriate temperature (Table 1
6
Evaluating Proinflammatory Cytokine Regulation by Donepezil and Rivastigmine in Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mRNA levels of proinflammatory cytokines (IL-6, IL-1β, COX-2, and iNOS) in BV2 microglial cells treated with donepezil or rivastigmine were evaluated by RT-PCR. Briefly, the cells (2.5 × 105 cells/mL in 12-well plates) were starved in serum-free media for 1 h before treatment with 200 ng/mL LPS or PBS for 30 min. The cells were then treated with drugs (donepezil, rivastigmine or donepezil + rivastigmine) or vehicle (1% DMSO) for 5.5 h. Total RNA extracted using TRIzol (Invitrogen, Carsbad, CA, USA) was used to synthesize cDNA via reverse transcription with the Superscript cDNA Premix Kit II (GeNetBio, Daejeon, Korea). PCR amplification was performed using 2xPrime Taq Premix (GeNetBio, Daejeon, Korea). The sequence information of the primers is shown in Table 1 .
7
PCR Amplification of DNA Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR amplification was done in a 20-µL reaction volume comprising 2 µL of template DNA (50 ng/µL), 1.0 µL (10 pmol) of each forward and reverse primer, 8 µL of 2x Prime Taq Premix (GENET BIO, Nonsan, South Korea), and 8 µL deionized distilled water. The reaction was incubated for initial denaturing at 94 °C for 5 min, followed by 32 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, with a final extension of 72 °C for 7 min. Amplified PCR products were subjected to agarose gel electrophoresis and they were visualized in a gel documentation system.
8
Genotyping of EGLN2 gene rs10680577 polymorphism
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We designed mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for genotyping of rs10680577 (4-bp ins/del) polymorphism within the promoter of EGLN2 gene. Mismatched C was introduced into the forward primers at -4 bp from the polymorphic site to create AleI restriction site. The forward and reverse primers were 5`-CCGTTATAAAAGATACTTATGTAAATCAC-3` and 5`-TTGGAATCAAGTGGCGTCG-3`, respectively.
Each 0.20 ml PCR reaction tube consisted of 1 μl of genomic DNA (~100 ng/ml), 1 μl of each primer (10 μM), 10 μl of 2X Prime Taq Premix (Genet Bio, Korea) and 7 μl ddH2O. The PCR conditions were 95 °C for 5 min, followed by 30 cycle of 30 s at 95°C, 30s at 57°C, and 30s at 72 °C, with a final extension step at 72 °C for 5 min. The PCR product (10 μl) was digested by AleI restriction enzyme (New England BioLabs, Beverly, MA). The digested products were electrophoresed on 2.5% agarose gel containing 0.5 μg/mL ethidium bromide, visualized on a UV transilluminator and photograph was takenFigure 1 . The del allele digested and produced 224 and 31 bp fragments while the ins allele undigested (259 bp). For the quality control of genotyping, approximately 20% of the random samples were regenotyped and the reproducibility was 100%.
Each 0.20 ml PCR reaction tube consisted of 1 μl of genomic DNA (~100 ng/ml), 1 μl of each primer (10 μM), 10 μl of 2X Prime Taq Premix (Genet Bio, Korea) and 7 μl ddH2O. The PCR conditions were 95 °C for 5 min, followed by 30 cycle of 30 s at 95°C, 30s at 57°C, and 30s at 72 °C, with a final extension step at 72 °C for 5 min. The PCR product (10 μl) was digested by AleI restriction enzyme (New England BioLabs, Beverly, MA). The digested products were electrophoresed on 2.5% agarose gel containing 0.5 μg/mL ethidium bromide, visualized on a UV transilluminator and photograph was taken
9
Actinobacteria DNA Extraction and PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA extractions for 87 isolates of Actinobacteria were performed as described by Hong et al. [1 ]. The primer pair 27F-1492R [1 , 28 ] was used for PCR amplification using the Kyratec PCR Supercycler (Kyratec, Australia). The PCR reaction mixture that consisted of 20–200 ng bacteria genomic DNA, 10.0 μL of 2X Prime Taq Premix (Genet Bio, Korea), 10 pmoles primer 27F and primer 1492R, and sterile ultrapure water was added to final volume of 20 μL. The cycling parameters were as described by Hong et al. [1 ].
10
Genotyping of Genetic Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genotyping of the variants were performed using PCR-RFLP or PCR method. The primer sequences are shown in Table 1. In each 0.20 ml PCR reaction tube, 1 µl of genomic DNA (~100 ng/µl), 1 µl of each primer (10 µM), and 10 µl of 2X Prime Taq Premix (Genet Bio, Korea) and 7 µl ddH 2 O were added.
Amplification was done with an initial denaturation step of 5 min at 95 °C followed by 30 cycles of 30 s at 95 °C, annealing at 68 °C for AGT rs699, 60 °C for AT1R
Amplification was done with an initial denaturation step of 5 min at 95 °C followed by 30 cycles of 30 s at 95 °C, annealing at 68 °C for AGT rs699, 60 °C for AT1R
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!