{"product_id":"neb-m1284l","title":"New England Biolabs, M1284L, RNase 4","description":"RNase 4 is a single-stranded endoribonuclease that cleaves RNA at uridine-purine (U\/R) dinucleotide sites.  \u003cb\u003eRelated Categories\u003c\/b\u003e RNases  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e One unit of RNase 4 is defined as the amount of enzyme required to cleave 1.8 pmol of a 45-mer RNA substrate containing a single U\/A cut site in 60 minutes at 25°C.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X NEBuffer™ r1.1 Incubate at 37°C 1X NEBuffer™ r1.1 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 100 µg\/ml Recombinant Albumin (pH 7 @ 25°C)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 100 mM NaCl 50 mM NaOAc 200 µg\/ml Recombinant Albumin 50% Glycerol pH 6 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e No  \u003cb\u003eFAQ\u003c\/b\u003e Q: Does RNase 4 (NEB #M1284) cut RNA at sites other than U\/A and U\/G? A: The observed preference of RNase 4 is U\/A \u0026gt; U\/G \u0026gt;\u0026gt;\u0026gt; C\/A. Low occurrence C\/A activity is observed when there are high concentrations of RNase 4 relative to the target RNA. Q: How much RNase 4 (NEB #M1284) should I use to digest RNA for LC-MS\/MS nucleotide sequence mapping? A: The addition of 1 µl of RNase 4 (NEB #M1284) (50,000 U\/ml) yields maximal sequence coverage for 10 µg of unmodified IVT RNAs 1-5 kb in length in a 30 µl reaction (1X NEBuffer r1.1, 1 M Urea) incubated at 37°C for 1 h. However, similar to using RNase T1 or bovine pancreatic RNase A, the ideal number of RNase 4 units needed can be higher or lower based on the target RNA. RNase 4 can be diluted in 1X NEBuffer r1.1, included as a 10X solution. Q: What is the chemical identity of 5´ and 3´ oligonucleotides generated after RNase 4 (NEB #M1284) cleavage? A: Following RNase 4 cleavage, upstream 5´ oligonucleotides will end with uridine and contain a mixed population of 3´-termini, mostly 3´-phosphate or 2´, 3´-cyclic phosphate. Downstream, 3´ oligonucleotides will begin with a purine (A or G) and contain 5´-hydroxylated termini. Q: How much RNase 4 (NEB #M1284) should I use for DNA-probe directed 5´-cap mRNA analysis? A: The current recommendation is to dilute RNase 4 (50,000 U\/ml) 1:30 in 1X NEBuffer r1.1. Adding 1 µl of diluted RNase 4 will be sufficient in reactions that contain 5 µg target RNA hybridized with 40 pmol of DNA probe (2023 Wolf et al. ACS Pharmacol. Transl. Sci.). Q: Can RNase 4 (NEB #M1284) be inactivated? A: Addition of 40 U of Murine RNase Inhibitor (NEB #M0314) or human placenta RNase Inhibitor (NEB #M0307) at room temperature effectively inhibits RNase 4 after standard digest conditions. For downstream applications that are not sensitive to detergents or chelating agents, a solution of 0.2 % (w\/v) SDS and 20 mM EDTA pH 8.0 completely inactivates RNase 4. Q: Is RNase 4 (NEB #M1284) endonucleolytic activity sensitive to RNA modifications? A: Many uridine modifications have been tested with zero to minimal decrease in RNase 4 activity, including pseudo-, N1-methyl-pseudo-, 5-methoxy, and dihydrouridine species (Ψ, m1Ψ, mo5U, and D). To efficiently digest RNAs containing these modifications, especially m1Ψ, we recommend increasing the added volume from 1 µl to 3 µl of RNase 4 (50,000 U\/ml) in equivalent 10 µg RNA digests. Since RNase 4 endonucleolytic activity requires the 2´-hydroxyl of the uridine nucleotide, modifications such as 2´ -O methyluridine (Um) completely inhibit RNase 4 activity. Q: Does RNA secondary structure impact RNase 4 (NEB #M1288) activity? A: For optimal nucleotide sequence mapping coverage, it is recommended to perform RNase 4 digests under denaturing conditions to limit the possible influence of RNA secondary structure. A digest reaction containing a final urea concentration at 1 M, incubated at 37 °C for 1 h, will ensure most RNA is available for endonucleolytic cleavage. RNase 4 endoribonuclease activity tolerates up to 3 M urea or 50% formamide (v\/v). T4 Polynucleotide Kinase end repair activity tolerates up to 3 M urea or 25 % formamide (v\/v). Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Q: Is magnesium required for RNase 4 activity? A: RNase 4 does not require magnesium for endoribonuclease activity. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835483869353,"sku":"M1284L","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m1284l","provider":"Iright","version":"1.0","type":"link"}