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Southern Blotting: Hybridization, Washes, and Development

来源:生物谷  2007/7/5 22:39:00   访问量:5538
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A. Hybridization

1. Denature probe (dig-dUTP labeled) for 5' at 90-100° C in boiling water bath. [Perform this step for newly labelled probes (in eppies) or "used" probes already in hyb solution (stored at -20° C in 50 ml conicals).]

2. At the same time, prehyb blot with at least 15 ml hyb solution in a hyb tube for at least 5'. Any prehyb time of 5' or longer is adequate.

3. If using a newly labelled probe, pipet probe into hyb solution (not onto the blot). If reusing a probe, pour off the (pre)hyb solution, save at 4° C for reuse; then dump the used probe/hyb solution onto the blot. Note: For newly labelled probes, try 10-15 µl of labeled DNA to 15 ml hyb solution for hybridization.

4. Incubate blot for at least 5 hours at 42° C...usually O/N.

B. Washes and Immunochemiluminescent Detection

1. Pour off probe and save at -20° C in 50 ml conical tube.

2. Wash blot with Blot Wash #1: 2 x 5' at room temp (RT).

3. Wash with Blot Wash #2: 2 x 15' at 55° C.

4. Wash with Buffer 1: briefly (1') at RT.

5. (Optional; see above) Block with Buffer 2: 30' (O/N okay) Save and store at 4° C.

6. Incubate with antibody-conjugate for 30' (save at 4° C).

7. Wash with Buffer 1 to remove unbound antibody-conjugate by washing 2 x 15' with Buffer 1.

8. Equilibrate blot with Buffer 3.

C. Visualization

1. Prewarm CSPD Ready-to-Use to RT.

2. Transfer blot carefully from Buffer 3 (let drip ~5'') to a plastic tray.

3. In the darkroom, incubate the blot with ~25 ml CSPD Ready-to-Use for ~1'; dump sol'n back into a dark bottle.

4. Drag blot along the edge of the tray to remove excess liquid, and place blot onto a plastic report cover.

5. Blot around edges with a tissue and then "close" the plastic cover. Rub gently to remove bubbles.

6. Expose to film. Incubate cassette at 37°C for 10 minutes, then return to RT. Exposures vary from blot to blot (1' to 2 hr).

Note: CDP Star is another detection reagent from Boehringer-Mannheim (that may be used instead of CSPD Ready-to-Use). It is supposed to be ~10X more sensitive.


Stripping Southerns

1. Wash blot in water for 2-5'.

2. Strip with 500 ml stripping solution (0.2N NaOH 0.1% SDS - made fresh) at 37 deg C for 30'.

3. Wash with 2x SSC at RT/1'. Keep in SSC until ready to use.

Prehyb ~5' and hybridize to next probe as shown on Southern protocol.

Note: This solution should not be used with Northerns because of the NaOH.

 

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