Seven days after SNL or sham surgery, L5 DRG on the ipsilateral side was collected for DNA extraction. The treatment of genomic DNA with bisulfite was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA), according to the manufacturer's instructions. The region of the Kcna2 gene promotor and 5′UTR from the −540 to +500 bp site consisted of 65 CpG sites and were amplified. For the pyrosequence study, 4 pairs of primers of Kcna2 modified with 5′-Biotin (Supplementary Table 3 ) were used to amplify the bisulfite DNA. The master mix of the binding buffer, streptavidin-sepharose beads, and PCR products were then prepared for the binding reaction in a 96-well plate. The pyrosequence was performed using a PSQ HS96 (Biotage, Charlotte, NC) to determine percentage methylation at each CpG site. For the clone-sequencing study, the same primers without biotin modification were used in PCR amplification. The PCR products were purified and subcloned into pMD T-19 (Takara). After an overnight bacterial culture, 20 subclones from each PCR assay were subjected to direct sequencing.
Pmd t 19
Manufactured by Takara Bio
The PMD T-19 is a thermal cycler designed for PCR (Polymerase Chain Reaction) amplification of DNA samples. It features a compact and durable construction, with a temperature range of 4°C to 99°C and a thermal ramp rate of up to 5°C per second. The PMD T-19 can accommodate standard 0.2 mL PCR tubes or 96-well microplates, providing a reliable and efficient platform for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Psq hs96, by Biotage (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Epitaq hs polymerase, by Takara Bio (1 mentions)
Ez dna methylation gold kittm, by Zymo Research (1 mentions)
3 protocols using pmd t 19
1
DNA Methylation Analysis of Kcna2 Gene
2
Genome-Wide DNA Methylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
gDNA isolated from both F2 thoracic aortas and F1 sperm was carried out by using QIAamp DNA Mini Kit (QIAGEN, 51304, Hilden, Germany). Bisulfate treatment of gDNA was performed by using EZ DNA Methylation-Gold KitTM (ZYMO Research, D5005, Irvine, CA, USA), according to the manufacturer’s instructions. PCR primers were designed with MethPrimer (http://www.urogene.org/methprimer/ ), and bisulfite-treated DNA was amplified with EpiTaq HS polymerase (Takara, R110Q, Beijing, China).
The PCR products were separated by using agarose gel electrophoresis and purified by using the Wizard® SV Gel and PCR Clean-Up System (Promega, A9281, Madison, WI, USA). Purified DNA fragments were cloned into pMD T-19 (Takara, No. 6013Code) and ten clones from each PCR assay were subjected to next direct sequencing. The final sequence results were analyzed by online QUMA (quantification tool for methylation analysis) software [22 (link)]. Primer sets used in bisulfate modified PCR are shown in Supplementary TableS1 .
The PCR products were separated by using agarose gel electrophoresis and purified by using the Wizard® SV Gel and PCR Clean-Up System (Promega, A9281, Madison, WI, USA). Purified DNA fragments were cloned into pMD T-19 (Takara, No. 6013Code) and ten clones from each PCR assay were subjected to next direct sequencing. The final sequence results were analyzed by online QUMA (quantification tool for methylation analysis) software [22 (link)]. Primer sets used in bisulfate modified PCR are shown in Supplementary Table
3
Cloning and Characterization of Antioxidant Enzyme in Shrimp
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length cytMnSOD of M. nipponense was obtained with rapid amplification of cDNA ends (RACE) methodology using hepatopancreas cDNA as the template. To obtain partial cDNA sequences, two degenerate primers, P1 (5'-gttYKccYatatcaatgctg-3') and P2 (5'-cKRaggttcttgtactgaMgg-3'), were designed based on the highly conserved nucleotides of known cytMnSOD of arthropods. According to the above cDNA sequences, specific primers (5'-Race outer primer-5'-ATGTGTAAGCTGAAGGGGGTCC-3', 5'-Race inner primer-5'-CAAGCCAGCCCCAACCAGAA-3'; 3'-Race outer primer-5'-TCACGTTCTTCCTCCCCTG A-3', 3'-Race inner primer-5'-TTCTGGTTGGGGCTGGCTTG-3') were designed to characterize the 5'-and 3'-regions of the cytMnSOD cDNA by RACE-PCR (TaKaRa) according to the manufacturer protocol. The full-length PCR product was cloned into pMDT-19 (TaKaRa) and sequenced from both directions by a commercial sequencing company (Invitrogen). Finally, the full-length SOD cDNA fragment of M. nipponense was obtained by overlapping three cDNA fragment sequences.
Sequences were compared and analyzed with the BLAST algorithm at the National Center for Biotechnology Information (NCBI). Potential N-glycosylation sites and signal peptide were predicted by NetNGlyc 1.0 Server (http://www.cbs.dtu. dk/services/NetNGlyc/) and SignalP program, respectively.
Sequences were compared and analyzed with the BLAST algorithm at the National Center for Biotechnology Information (NCBI). Potential N-glycosylation sites and signal peptide were predicted by NetNGlyc 1.0 Server (http://www.cbs.dtu. dk/services/NetNGlyc/) and SignalP program, respectively.
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!