{"product_id":"neb-m1288l","title":"New England Biolabs, M1288L, RNase 4 Digestion and 3´ End Repair Mix","description":"  \u003cb\u003eRelated Categories\u003c\/b\u003e RNases,, RNA Modification  \u003cb\u003eSpecification\u003c\/b\u003e \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\u003eFAQ\u003c\/b\u003e Q: How much RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) should I use to digest RNA for LC-MS\/MS nucleotide sequence mapping? A: The addition of 1 µl of RNase 4 Digestion and 3′ End Repair Mix yields maximal sequence coverage for up to 20 µg of unmodified in-vitro transcribed RNA 1,000-9,000 nucleotides in length, in a 30 µl reaction (1X NEBuffer r1.1, 1 M Urea) incubated at 37°C for 1 h. Similar to other RNases used for LC–MS\/MS sequence mapping, the amount of RNase 4 to include for maximal sequence coverage requires optimization. Generally, longer and more modified RNA will require the addition of more RNase 4 Digestion and 3′ End Repair Mix to obtain the highest sequence coverage. For modified RNA substrates we recommend first increasing the amount of RNase 4 Digestion and 3′ End Repair mix from 1 µl to 3 µl. 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: Can I use RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) for DNA-probe directed 5′ cap mRNA analysis? A: We recommend dilution of RNase 4 (NEB #M1284) in our standard protocol for DNA-probe directed 5′ cap mRNA analysis. Implementation of this protocol with RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) will result in concurrent dilution of T4 PNK thereby impairing the efficiency of 3’-end repair. Therefore, we currently recommend using standalone RNase 4 (NEB #M1284) for DNA-probe directed 5’ cap mRNA analysis. 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. Q: Is RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) sensitive to RNA modifications? A: Many uridine modifications have been tested with minimal decrease in RNase 4 and T4 PNK 3′ activities, 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. Since RNase 4 endonucleolytic activity requires the 2′-hydroxyl of the uridine nucleotide, modifications such as 2′-O methyluridine (Um) at the cleavage site 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: Does RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) require magnesium? A: We recommend use of included NEBuffer r1.1 for RNA digestions with RNase 4 Digestion and 3′ End Repair Mix following our recommended digestion protocol. The 3′ end repair activity of T4 polynucleotide kinase requires magnesium for efficient phosphate removal. However, it is noteworthy that like RNase A, RNase 4 does not require magnesium for endoribonuclease activity. 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. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835478364329,"sku":"M1288L","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m1288l","provider":"Iright","version":"1.0","type":"link"}