M T3 T7

Fig. 2. Gel electrophoresis of in vitro transcribed sense and antisense riboprobes for the Drosophila melanogaster u-shaped gene. 1% agarose gel in TAE-buffer. Lane M: 1-kb ladder. Lane T3: digoxigenin (DIG)-labeled sense probe transcribed with T3-RNA polymerase; note the smear. Lane T7: DIG-labeled antisense probe transcribed with T7-RNA polymerase; note the strong band at about 1.6 kb. The clone of u-shaped cDNA was kindly provided by Marc Haenlin.

Fig. 2. Gel electrophoresis of in vitro transcribed sense and antisense riboprobes for the Drosophila melanogaster u-shaped gene. 1% agarose gel in TAE-buffer. Lane M: 1-kb ladder. Lane T3: digoxigenin (DIG)-labeled sense probe transcribed with T3-RNA polymerase; note the smear. Lane T7: DIG-labeled antisense probe transcribed with T7-RNA polymerase; note the strong band at about 1.6 kb. The clone of u-shaped cDNA was kindly provided by Marc Haenlin.

f. 40 U of T7-, T3- or SP6-RNA polymerase (see Note 17).

g. DEPC-treated H2O to a final volume of 20 ^L.

2. Mix and incubate for 2 h at 37°C (see Note 18).

3. Add 1 ^L of RNase-free DNase and incubate for additional 30 min at 37°C to remove template DNA.

4. Stop the reaction by adding 2 ^L of 0.2 MEDTA, pH 8.0.

5. Add 2.5 ^L of 4 M LiCl and 75 ^L of -20°C 100% ethanol, mix well, and precipitate for 30 min at -80°C or for 2 h at -20°C.

6. Spin in a microcentrifuge at maximum speed for 15 min at 4°C.

7. Remove the supernatant and wash the pellet with -20°C 75% ethanol.

8. Spin for 5 min at maximum speed at 4°C, remove the ethanol, and air-dry.

9. Resuspend the pellet in a small amount of DEPC-treated H2O in case you want to perform a gel electrophoresis (step 10), or in 100 ^L of HB. If the pellet does not dissolve, incubate at 56°C (see Note 19).

10. Check amount, quality, and dimension of RNA transcripts on a standard agarose gel (see Fig. 2 and Note 20).

Although ethanol precipitation is usually the method of choice for cleaning and concentrating riboprobes, sometimes it is not efficient in removing unused labeled nucleotides, which consequently cause high background. The protocol below employs NucTrap push columns (Stratagene; see also ref. 4) to specifically remove these contaminating nucleotides by gel filtration.

1. At the end of template digestion (Subheading 3.1.2., step 4) add 40 ^L of DEPC-treated H2O to the reaction.

2. Equilibrate the column with 80 ^L of STE buffer.

3. Apply the riboprobe solution (60 ^L) to the column.

4. Elute the riboprobe with additional 70 ^L of STE buffer; the eluted volume is about 110 ^L.

5. Precipitate the riboprobe with 0.5 vol of 7.8 M ammonium acetate and 3 vol of 100% ethanol at -20°C for 30 min.

6. Spin in a microcentrifuge at maximum speed for 15 min at 4°C.

8. Spin for 5 min at maximum speed at 4°C, remove the ethanol, and air-dry.

9. Dissolve the pellet in 100 ^L of HB.

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