Transfer Preparation

1. Prepare the gel for transfer by soaking it for two 20-minute periods in 10x SSPE at room temperature with gentle shaking.
2. During the gel washing procedure, prewet the membrane in distilled water for 5 minutes followed by a 5-minute soak in 10x SSPE.
3. Transfer the RNA in 10x SSPE by capillary action using a sponge to enhance capillary action (we found that not using a sponge worked just as well).
4. Fix the RNA to the membrane by baking for 2 h at 80℃ or by placing the nylon membrane, with the RNA-side down, on a short wave (254 nm or 302 nm) transilluminator for 5 minutes.

Comments

1. If a thick gel or a high concentration of agarose (>1.3%) has been used, it is beneficial to allow the gel to soak for 10-20 minutes in 0.05 M NaOH made up in 1x SSPE. In this latter case, the two 10x SSPE washes are carried out after the NaOH wash. This mild NaOH treatment increases transfer efficiency without greatly degrading the RNA.
2. The authors recommend using a charge-modified nylon membrane because it has a higher binding capacity than nitrocellulose and will withstand multiple stripping and reprobing. We found that Hybond N⁺ work really well.
3. The RNA bound to the membrane can be viewed for transfer efficiency or photographed under UV transillumination: no staining is required.

Hybridization and Autoradiography

1. Prehybridize overnight and hybridize for 12-36 h at 42℃with gentle shaking.
2. Wash the membranes: two 20-minute washes in 2x SSC, 1% SDS at room temperature followed by two 20-minute washes in 1x SSC, 0.5% SDS at 50-55oC.
3. Autoradiograph with Kodak XAR film at -70℃ for 2-3 days using Dupont Cronex Lightning Plus screens.

Comments

1. We routinely use the following prehybridization and hybridization solution: 50% deionized formamide, 0.47x Denharts solution, 4.7x SSPE, 0.1% SDS, 0.18 mg/ml denatured salmon sperm carrier DNA, and 5% dextran sulfate. Note that the addition of fat-free mild powder to the prehybridization solution (0.34% final concentration) will decrease the background on membranes which have higher binding capacities.
2. We use DNA probes labeled to high specific activity (>1x10⁸cpm/ug) using [a-³²P]CTP by the random primer labeling procedure (1).

(1) Feinberg, A.P. and Vogelstein, B. 1983. Anal. Biochem. 132, 6.

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