Bulk synthetic RNA preparation

Saturday, April 30, 2005

Description
A method to prepare large ammounts or synthetic RNA using T7, T3 or SP6 RNA polymerases.

Procedure
Template Preparation and RNA Transcription

PCR generated templates or 2 x Cesium Cl2 banded plasmid DNA with inserts ranging in size from 200 bp & up can be used for transcription rxns. pBluescript contains promoter sequences specific for T7/T3 RNA polymerases, pSP64 and 65 contain SP6 and T7 (Melton et al. 1984).

As an alternative to 2x Cesium Cl2 banding, a good Plasmid purification kit can be used (Eppendorf Perfectprep):

- Make as usual per Alkali plasmid prep, starting with 50 ml culture. Resuspend in 300 ul TE.
- Add 300 ul ice cold 5M LiCl. Mix well, spin for 10'' in microfuge at 4oC (presipitates some RNA).
- Transfer supernatent to fresh tube; add 600 ul isopropanol. Mix well, centrifuge 5'' at RT.
- Remove all supernatent; rinse pellet with 70% EtOH; dry carefully.
- Resuspend in 250 ul TE containing 20 ug/ml RNAse A. Incubate 30'', RT.
- Add 250 ul of 1.6 M NaCl containing 13% (w/v) PEG 8000. Mix well. Centrifuge 5'' at 4oC.
- Remove sup completely; dissolve pellet in 200 ul TE.
- Extract once with phenol, once with phenol,/ CHCl3, and once with CHCl3.
- Add 1/2 volume of RNAse-free 7.5 M NH4OAc and 2 volumes of RNAse-free ethanol.
- Mix, store at RT for 5''. Spin for 5'' at RT. Rinse with 70% EtOH, dry well.
- Redissolve in 100 ul RNAse free TE. Check concentration by O.D. and gel electrophoresis.

1. Linearize 25 ug of the template DNA with an enzyme that cleaves downstream from the polymerase site and insert in the multiple cloning region. Use enzymes that leave either blunt or 5'' protruding ends to alleviate 3'' nonspecific initiation of transcription. Treat the digestion reaction with 50 mg/ml Prot. K for 30'' at 37o, followed by 2x phenol/chloroform extractions and EtOH precipitation. Resuspend template DNA in 25 ml TE, pH 8.0 for a final concentration of approx. 1mg/ml. PCR generated templates: use 200-300ng DNA that has been phenol/chloroform extracted & EtOH precipitated.
Note: template must be RNase free. Also, use, RNase-free tubes (Eppendorf)

Transcription reaction - scale up as needed
Mix at r.t. in the following order:

DEPC-water 7 ul
5X Buffer 4
0.1M DTT 2
10 mM UTP 1
10 mM ATP 1
10 mM CTP 1
10 mM methyl - GTP 1
2.5 mM GTP 1
Restricted DNA (1mg/ml) 1
RNasin (40 U/ul) 0.5
Polymerase (20U/ml) 0.5
incubate 37o for 1 hr

- Add 10 more units of RNA polymerase and incubate 37o for another hour.

Note - lowering the incubation temperature can increase the amount of full length product (Krieg and Melton (1984) NAR 12-7057).

3. DNase template:
Add 1.0 ul DNase (RNase-free) and incubate at 37o for 10''.

4. Increase volume (if necessary) to 100 ul and extract with phenol/CHCl3 and then CHCl3. Precipitate with NaOAc and resuspend in 100 ul water.

5. Pass over RNA spin column after rinsing column twice (at least) with water. Precipitate recovered effluent with NaOAc and resuspend in water.

Recipes

Supplies
T7/ T3/ SP6 RNA Transcription Kit, NTP''s, DTT (Epicentre Technologies; enzymes are cheapest)
Methyl (capped)-G: GE Healthcare 27-4635. Dissolve in DEPC-treated water to bring to 10 mM.
Eppendorf Prime RNase Inhibitor
RNase free DNase - Any good manufacturer
DEPC-treated water and 3 M NaOAc
RNAse-free 80% and 100% ethanol
Phenol, Chloroform
RNA Sephadex-G50 spin columns
Filter-pluggeg pipet tips
RNAse-free microfuge tubes (Eppendorf)

Tips

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Bulk synthetic RNA preparation