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PvuRts1l restriction enzyme
PvuRts1I is a restriction enzyme that selectively cleaves 5-hmC-containing DNA sequences. PvuRts1I is able not only to discriminate between 5-hmC and 5-mC but it can also cleave both glucosylated and nonglucosylated 5-hmC DNA templates.
|PvuRts1l restriction enzyme||AF-113-0100||100 units/ 100 µl||95 $|
- Isolated from a recombinant source
- Selectively cleaves 5-hmC-containing DNA sequences
- cleave both glucosylated and non-glucosylated 5-hmC DNA sequences
- Supplied with optimized 10X Reaction Buffer
• Can be used to assess the global abundance of 5-hmC in genomic DNA 
- Ideal to control hMeDIP experiments
500 ng of hydroxymethylated, methylated and unmethylated DNA standards (Cat. No. AF-101-002) were incubated in the absence or presence of 1 unit PvuRts1l enzyme for 30 minutes at 32°C. Each sample was run on a 1.5% agarose gel with a 100 bp DNA ladder.
Mouse embryonic stem cell DNA was fragmented by sonication using the Diagenode Bioruptor® (size range: 100-500 bp). One µg of sheared mouse embryonic stem cells was mixed with the three DNA controls (C, 5-mC and 5-hmC) from the hMeDIP kit and incubated with (PvuRts 1 treatment) or without (NT) 1 unit PvuRts1I restriction enzyme for 30 minutes at 32 °C. After purification with the PrepEaseTM PCR Purification 96-well Plate kit (PN 78761, USB®), this reaction was recovered in 108 µl nuclease-free water and added to 12 µl buffer H1 from the hMeDIP kit (Cat. No. AF-104-0016). The IP incubation mix was then heat denatured and hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit (Cat. No. AF-104-0016). This kit includes: the monoclonal rat antibody & Rat IgG (Cat. No. AF-105-0025). Finally qPCR using specific primer pairs for the unmethylated, methylated and hydroxymethylated DNA sequences were performed. After PvuRts1I enzymatic treatment, the recovery of the 5-hmC control is almost completely abolished.
Source: Purified from an E. coli strain, which carries a PvuRts1I gene construct.
Storage conditions: Storage at -20°C is recommended. Since the 10x reaction buffer contains DTT, it is highly recommended to store it in small aliquotes at -20°C. For long-term storage, store both the enzyme and the 10 reaction buffer at -80°C. Avoid multiple freeze thawing. When stored properly the enzyme is stable for at least 6 months from date of receipt.
• 1 tube of 100μl of PvuRts1I enzyme
• 1 tube of 300μl of 10 X PvuRts1I reaction buffer (enough for 120 x 25μl assays)
Cleaves 5-hmC containing DNA sequences (cleaves also glucosylated 5-hmC)
Ideal to control hMeDIP and dot blot experiments