Genomic DNA was extracted from 200 mites from each T. urticae strain using the G-spin™ Total DNA Extraction Mini Kit (Intron, Seongnam, Korea) according to the manufacturer’s instructions. Approximately 100 ng of DNA was used as template DNA for PCR. The reactions were performed using a PCR Premix kit (HotStart, Bioneer Co., Daejeon, Korea) and the primers listed in Table 2 . The resulting PCR products were purified and directly sequenced using Bioneer Co. The pyrosequencing protocol consisted of 45 PCR cycles performed with the forward primer and biotinylated reverse primer at 0.5 μM, each in 20 μL reaction mixture containing 1× Taq enzyme reaction mix (Enzynomics, Daejeon, Korea). The following cycling conditions were used: one cycle at 95 °C for 15 min; 45 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; and a final step at 72 °C for 10 min. The reactions were performed using a PyroGold reagent kit and a PyroMark ID system (Qiagen, Germantown, MD, USA).
Pcr premix kit
Manufactured by Bioneer
The PCR Premix kit is a pre-formulated solution containing all the essential components required for performing Polymerase Chain Reaction (PCR) analysis. The kit includes a thermostable DNA polymerase enzyme, nucleotides, buffer, and other necessary reagents, pre-mixed in an optimized formulation to simplify and streamline the PCR setup process.
Automatically generated - may contain errors
Lab products found in correlation
Primscript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Trizol, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pyromark id system, by Qiagen (1 mentions)
Pyro gold reagent kit, by Qiagen (1 mentions)
Improm 2 reverse transcription system kit, by Promega (1 mentions)
Rt premix kit, by Bioneer (1 mentions)
High pure rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
6 protocols using pcr premix kit
1
Genomic DNA Extraction and Sequencing of T. urticae
2
Lung Tissue RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After homogenizing the lung tissue, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) and then converted to cDNA using the ImProm-II Reverse Transcription System kit (Promega Corporation, Fitchburg, WI, USA). PCR amplification was performed using the PCR PreMix kit (Bioneer, Daejeon, Korea) with the addition of sense primers (20 pmole/mL) and antisense primers (20 pmole/mL). The primer sequences are shown in Table 2 . The PCR products were electrophoresed on a 1.2% agarose gel.
3
Sensitive miRNA Detection via Nicking Isothermal Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two reaction mixtures were prepared separately on ice. The first mixture contained RNA sample, external control miRNA (mir-159a), Buffer R, dNTPs, stem-loop probes, and amplifiers. The second mixture contained Bsm DNA polymerase and Nb.Bpu10I nicking endonuclease. The two mixtures were combined at 37 °C and incubated for 3 h. The final reaction mixture contained 20 μL of varying amounts of synthetic miRNA or 800 ng of total RNA, 20 fmol of external control miRNA, 60 fmol of stem loop probes, 30 fmol of amplifier, 0.5 U of Bsm DNA polymerase, 7 U of Nb.Bpu10I, 1× Buffer R, and 250 μM dNTPs. Subsequent linear amplification was performed in 20-μL reaction volumes containing PCR Premix kit (Bioneer, Daejeon, Korea), 2 μL of miRNA assay reagent, and 20 fmol of primer (5′-FAM-GTGCCAGCAAGATCCAATCTAGA-3′) to label the amplified products. Reactions were carried out with the following amplification profile: initial denaturation at 95 °C for 5 min; 20 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s; and final extension at 72 °C for 7 min.
4
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using the High Pure RNA Isolation Kit (Invitrogen Co., Waltham, MA, USA) according to the manufacturer's instructions and quantified using a NanoDrop (Thermo Scientific, Waltham, MA, USA). For the reverse transcription reaction, 1 μg of isolated RNA and 1 μL of oligo DT (100 pM) were combined with DEPC to a total volume of 11 μL, followed by the reverse transcription reaction using the RT Premix Kit (Bioneer Co., Daejeon, Republic of Korea) as per the manufacturer's protocol. PCR for MITF, Tyrosinase, and MC1R genes was conducted using 2 μL of the cDNA products obtained from the reverse transcription reaction, with the PCR Premix Kit (Bioneer Co.) according to the manufacturer's instructions. The cDNA products amplified by reverse transcription and polymerase chain reaction were analyzed by electrophoresis on 2.0% agarose gels containing ethidium bromide. The primer sequences used for PCR are provided in Table 1.
5
Isolation and Analysis of Cell Types from Corpus Luteum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cultivated GCs from follicles were collected using Trizol (Takara, Shiga, Japan) and LCs (Figure 1 b), LTCs (Figure 1 g), and LECs (Figure 1 f, yellow line area) from secretion-phase CLs were also dissociated using Trizol (Takara) from culture dishes. The part of the secretion-phase CLs, which were used to isolate LTCs, were gathered into Trizol (Takara) and these samples were used as positive control in the experimental steroidogenic verification. All processes of the extraction mRNA using Trizol (Takara) were followed according to product manual. The mRNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher). The mRNA was extracted using Trizol, then total 5.0 μg mRNA was transcripted to cDNA using PrimScript 1st strand cDNA synthesis kit (Takara), and reverse transcription was performed at 45 °C for 60 min after 95 °C for 5 min. The 1.0 μL synthesized cDNA were used to conduct PCR according to the primer conditions (Table 1 ) using PCR premix kit (Bioneer, Seoul, Republic of Korea). The PCR products were separated with 2.0% aga-rose gel electrophoresis at 100 V for 20 min and visualized with ethidium bromide (Sigma) and UV light. The PCR product expression was analyzed with ImageJ software (NCBI, USA).
6
mRNA Extraction and Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mRNA was extracted using TRIzol (Takara, Shiga, Japan), and concentration was measured using NanoDrop 2000 spectrophotometer (Thermo Scientific). Total 5.0 μg mRNA was used to synthesis cDNA using PrimScript 1 st strand cDNA synthesis kit (Takara), and reverse transcription was performed at 45℃ for 60 min after 95℃ for 5 min.
The 1.0 μL synthesized cDNA were used to conduct PCR and was performed according to the primer conditions (Supplementary Table S2) using PCR premix kit (Bioneer). Then, the products were separated with 2.0% agarose gel electrophoresis at 100 V for 20 min, stained with ethidium bromide, visualized with UV light, and mRNA expression was analyzed with ImageJ software (NCBI).
The 1.0 μL synthesized cDNA were used to conduct PCR and was performed according to the primer conditions (Supplementary Table S2) using PCR premix kit (Bioneer). Then, the products were separated with 2.0% agarose gel electrophoresis at 100 V for 20 min, stained with ethidium bromide, visualized with UV light, and mRNA expression was analyzed with ImageJ software (NCBI).
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!