Transfer
1. After electrophoresis, wash gel in 20X SSC for 20 minutes.
2. Pre-wet membrane in Milli Q water or in 2X SSC.
3. Transfer gel using Vacugene apparatus. The order of components, starting from the bottom, is: screen, membrane, mask, gel. The membrane should be ~ 1 cm larger than the opening of the mask all around. Fill with 20X SSC (10X SSC works equally well) until gel is covered. Transfer at 70—80 cm/H₂O pressure for 2 hours.
4. After transfer, rinse membrane in 2X SSC and crosslink (“Auto Cross Link” function of the Stratalinker).

Prehybridization
Pre-hyb solution (final concentrations):                  Volume of stock solutions required:
55% formamide                                                        5.5 ml formamide
5X SSC                                                                     2.5 ml 20X SSC
1x Denhardts                                                            200 μl 50X Denhardts
50mM phosphate buffer, pH 7.0                            2.5mL 0.2M phosphate buffer, pH7.0
250μg/mL ssDNA (fish sperm DNA)                     250 μl 10 mg/ml ss DNA

-Alternatively, 500μL 50X Denhardts and 1.5mL 0.2M phosphate buffer, pH 7.0 (other components the same) will work equally well.
-For 100mL 0.2M phosphate buffer (pH 7.0), mix 57.7mL 1M Na2HPO4 and 42.3mL 1M NaH₂PO₄
-Incubate the membrane in the pre-hyb solution for at least 4 hours at 42℃ (overnight, if preferred).

Hybridization
Hyb solution (final concentrations):                                            Volume of stock solutions required:

-For labeling and counting of the probe, see protocol for Southern blotting.
-Prepare hyb mixture without the probe. Place the membrane in the container; add the hyb solution, and finally, the probe. Incubate overnight at 42℃.

Washing
Two alternatives methods (work equally well):
Method 1
-Remove membrane from hyb solution and rinse 3x at room temperature with 2X SSC+0.1% SDS solution.
-Wash the membrane 2x 30 minutes, in 2X SSC+0.1% SDS, at 420C while shaking.
Method 2
-Remove membrane from hyb solution and rinse 3x at room temperature with 2X SSC+0.1% SDS solution.
-Wash the membrane 4x 5 minutes in 2X SSC+0.1% SDS at room temperature while shaking.
-Wash the membrane 2x 15 minutes in 0.1X SSC+0.1% SDS at 500C while shaking.
-Regardless of the method used, check counts with Geiger in between the longer washes, especially if the probe is not particularly “hot”. If the membrane is still “hot” overall, continue washing.Otherwise, stop and expose the membrane to a phosphorimager screen for at least 4 hours.

Stripping
-Place membrane in 0.1X SSC+0.1% SDS, 2x 30 minutes, at 850C with vigorous shaking.

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