HomeFAQsHow much RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) should I use to digest RNA for LC-MS/MS nucleotide sequence mapping?
FAQ: 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?
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.
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