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The following protocol is for MEGAscript II Kit (Ambion).
1. PCR amplify the DNA template. The 5'-end of the template should contain the minimum promoter sequenences of T7 or Sp6 or T3.
A 5-primer with the minimum promoter sequences may be used for PCR. Agarose gel may be used to check that there is only one PCR product.
For plasmid template, the plasmid must be linearized first by restriction digestion 3' of the desired transcript end. Plasmid DNA may contain high level of RNase. After digestion, DNA may be treated with 0.1-0.2 mg/ml of proteinase K and 0.5% SDS for 30 minutes at 50 ℃, followed by phenol:chloroform extraction and ethanol precipitation or gel purified.
2. Make the following reaction mix (water and NTPs should be added before 5X reaction buffer).
| Nuclease-free ddH2O |
3 ml |
| 100 mM ATP |
2 ml |
| 100 mM CTP |
2 ml |
| 100 mM GTP |
2 ml |
| 100 mM UTP |
2 ml |
| 5X Reaction Buffer |
4 ml |
| a-32P-UTP |
1 ml |
| linear DNA template (0.5 mg/ml) |
2 ml |
| Enzyme mix |
2 ml |
| Total volume |
20 ml |
3. Mix and centrifuge briefly.
4. Incubate at 37 ℃ for 2-4 hours.
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