A simple and effective transcription method to reduce heterogeneity at the 3' end of RNA

Summary

Ribosyl C2'-methoxy modification of the nucleotides at the 2 5' ends. Amplification of DNA by PCR with modified primers still reduces N+l activity, and since PCR can produce templates several kb long, less heterogeneous RNA can be prepared.

Operation method

A simple and effective transcription method for reducing heterogeneity of RNA 3' ends

Materials and Instruments

8umol L Top strand 8umol L Bottom strand 5' end 2 nucleic anomalies for methoxyl modification
20X buffer NTP mixture lmol LNaOH polyethylene glycol 8000 0.5mol LMgCl2 1mol L monosodium glutamate T7RNA polymerase loading buffer

Move

I Materials and equipment

1) 8umol/L top chain

2)8umol/L bottom chain, 5' end 2 nucleic anomalies modified with methoxylate

3)20X buffer:800 mmol/LNaCl.20 mmol/L arginine, 100 mmol/LDTT, 0.2% TritonX-100(pH8.l)

4) NTP mixture, 80mol/L each, adjusted to pH 8.1 with 1mol/LNaOH.

5) lmol/L NaOH.

6) Polyethylene glycol 8000, 400 mg/ml.

7) 0. _5mol/LMgCl2

8) 1mol/L monosodium glutamate (pH 8.l)

9)T7 RNA polymerase.

10) Sample loading buffer: 8 mol/L urea, lmmol/L LEDTA, 0,1% toluene cyanine FF, 0.1% bromophenol blue.


Methods

1) Mix the following substances in a sterilized microcentrifuge tube:

8umol/L top chain (final concentration 400nmol/L) 1ul

8umol/L bottom chain (2 nucleosides at the 5' end) 1ul

Bottom chain (2 nucleosides at the 5' end 1ul acid-methoxy modified, final concentration 400nmol/L)

20X buffer 2ul

NTP mixture 2ul

Polyethylene Glycol 8000 5ul

0. _{5mol/LMgCl2 1} .12ul

1mol/L monosodium glutamate 1ul

T7RNA polymerase 1ul

Add nuclease free water to a total volume of 20ul

2) React at 37℃ for lh.

3) Add the sample solution, variant polyacrylamide gel electrophoresis purification and recovery, stored in a 20 ℃.

Caveat

1) The standard system is 20ul but the system can be scaled up for each component to increase the amount of transcripts2) In general, the number of moles of NTP is related to the amount of Mg2+.moles of NTP to Mg 2+ moles of Mg 2+ is 1:1.75, but this ratio may need to be optimized.3) For some templates, monosodium glutamate can improve the recovery of transcription products.

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