In Vitro Transcription with SP6 Polymerase

Monday, October 20, 2003

Description
All reagents and tubes should be RNase-free. Make solutions up in new plastic vessels using ddH2O. Autoclave the NaCl, MgCl2, Tris, glycerol, and gelatin solutions. Do not touch the undersides of tube caps or pipette tips with bare hands.

Procedure
1. Linearize the template DNA by restriction enzyme digestion.

2. Add an equal volume of NETS and then add twice that volume of phenol. Mix by inverting the tube several times and centrifuge at maximum speed for 2 min in a microcentrifuge to separate the phases. Recover the aqueous phase and add an equal volume of phenol, centrifuge again and recover the aqueous phase.

3. Add an equal volume of chloroform and mix by inverting the tube several times and centrifuge at max speed in a microcentrifuge to separate the phases. Recover the aqueous phase and add an equal volume of chloroform, centrifuge again and recover the aqueous phase.

4. Add 2.5 volumes of ethanol and chill on ice for 5 min to precipitate the DNA. Centrifuge at max speed in a microcentrifuge for 15 min at 4癈.

5. Remove the supernatant and resuspend the DNA pellet in ice cold 70% ethanol, 0.1 M NaCl and centrifuge at max speed in a microcentrifuge for 15 min.

6. Remove the supernatant and dissolve the DNA pellet in TE, add NaCl to 0.2 M and add 2.5 volumes of Ethanol. Chill on ice for 5 min to precipitate the DNA. Centrifuge at max speed in a microcentrifuge for 15 min at 4癈. Remove the supernatant and resuspend the DNA pellet in 70 % Ethanol, 0.1M NaCl and centrifuge at max speed in a microcentrifuge for 15 min.

7. Remove the supernatant and allow the pellet to air dry and then dissolve it in TE buffer to a concentration of 0.5 mg/ml.

8. Set up the transcription reaction with the following components: 10 靗 DNA, 10 靗 10X Transcription Buffer, 10 靗 10X nucleotides, 10 靗 5 mM m7GpppmG or GpppG, 2 靗 0.5 M DTT, 2 靗 RNasin, 5 靗 80 % glycerol, 5 靗 SP6 RNA polymerase, 46 靗 water (100 靗 total reaction volume).

9. Incubate reaction at 37癈 for 3 hr.

10. Add 5 靗 RQ1 RNase-free DNase and NaCl to 0.01 M and incubate at 37癈 for 1 hr (see Hint #1).

11. Run 3 靗 of the reaction on a 2 % agarose-TAE minigel to verify that the transcription and DNase treatment worked by looking for the appearance of an RNA transcript of appropriate size according to the Tobacco Mosaic Virus (TMV) RNA standard.

12. Add an equal volume of NETS and heat to 65癈 for 5 min and then precipitate the RNA as described for DNA in steps 2-7, with the exceptions of resuspending the RNA in water, not TE Buffer, and resuspend the final RNA pellet in 20 靗 of dd H2O.

13. Estimate the RNA concentration by running 1 靗 of the final solution on a gel next to the TMV RNA standard.

Recipes
0.5 M DTT

80 % (w/v) glycerol

Ethanol/salt 70 % Ethanol
0.1 M NaCl

NETS 0.2 M Tris-HCl, pH 8.0
20 mM EDTA, pH 8.0
2 % (w/v) SDS
0.3 M NaCl

5 mM m7GpppmG or GpppG Store at -70癈

Nucleotides (10X) 5 mM CTP
Store at -70癈
5 mM UTP
0.5 mM GTP
5 mM ATP

Transcription buffer (10X) 0.05 M DTT
1 mg/ml autoclaved Gelatin
0.08 M MgCl2
Store at -20癈
0.4 M Tris-HCl, pH 7.5

TAE 40 mM Tris:acetate
Working concentrations:
57.1 ml Glacial Acetic Acid
Add ddH2O to 1 liter
37.2 g disodium EDTA
242 g Tris base
pH 8.5
2 mM EDTA

TE Buffer 10 mM Tris
pH 8.0
1 mM EDTA

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In Vitro Transcription with SP6 Polymerase