Synthesize 5' capped transcripts

Summary

Some RNA transcripts require an m7 G(5')Ppp(5')G cap at the 5' end, and they have higher translational efficiency only in cell-free extracts or in African Xenopus oocytes. Transcripts with methylated caps have also been reported to have higher efficiency during in vitro shearing and greater resistance to nuclease in nuclear extracts

Operation method

Synthesis of 5' plus cap transcripts

Materials and Instruments

DEPC-treated water Transcription buffer lOOmmol LDTT RNasin rATP rCTP rUTP rGTP0.5 mmol L m7 G(5')ppp(5')G DNA template RNA sample buffer RNA load buffer MOPS buffer TE buffer

Move

I Materials and equipment

1) DEPC: Treated water

2) Transcription buffer (5X):200 mmol/LTris-HCI(pH7.9),30 mmol/LMgCl2,lOmmol/L. spermidine,50 mmol/LNaCl

3)lOOmmol/LDTT

4)RNasin

5)rATP, rCTP, rUTP, 2.5 mmol/L each

6)rGTP0.5 mmol/L

7) ^{m7 G} (5')ppp(5')G,5 mmol/L

8)DNA template, 1~2 mg/ml

9)RNA sample buffer: 10 mmoL/I. deionized formamide, 3.5 ml37% formaldehyde, 2 mlMOPS buffer
buffer

10) RNA loading buffer: 50% glycerol, lmmol/LEDTA, 0.4% bromophenol blue, lmg/ml ethidium bromide.

11)MOPS buffer: 0.2 mmol/LMOPS(pH7.0),50 mmol/L sodium acetate,5 mmol/LEDTA(pH8.0)

12)TE buffer


Methods

1) In vitro transcription to generate RNA

In a sterile centrifuge tube, add the following ingredients at room temperature.

5X Transcription Buffer 4ul

100 mmol/LDTT 2ul

RNasin 20U

rATP,rUTP,rCTP(2,5 mmol/L each) 4ul

GTP(O.5 mmol/L) 2ul

^m7 G(5')ppp(5')G(5 mmol/L) 2ul

DNA template (1~2 mg/ml) 1-2ug

Add nuclease-free water to final volume 19ul

2) Start the reaction by adding 1ul of SP6, T7 or T3 RNA polymerase (15~20U/ul).

3) Incubate at 37~40°C for 60 min.

4) Remove the DNA template as described for RNA standard transcription reaction and purify the RNA transcript.

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