Alkaline Southern Blot for Oligos
1.) Digest desired amount of DNA and run agarose gel.
2.) Photograph gel with a ruler next to markers. Then cut off left upper corner.
3.) Set up standard blotting-chamber using 0.4 N NaOH as transfer buffer (ca. 1 liter).
Pour into tray half full.
4.) Soak 4 pieces of Whatman-paper, cut to stone block size, in ddH2O and lay on block. Role off airbubbles with pipette.
5.) Place gel upside down on Whatman/stone block. Wet gel with 0.4 N NaOH.
6.) Underlay geledges with small stripes of parafilm. Avoid ares, where DNA is present.
7.) Cut Zeta probe filter to gel size and soak in ddH2O. Carefully place on top of gel.
8.) Fill separat glassrack with rest of 0.4 N NaOH and soak 2 Whatman paper (gel size).
Then place on top of gel. Roll off all airbubbles.
9.) Cut white papertowels to gel size ca. 8 cm of height and place on top.
10.) Leave O/N.
11.) In am remove the filter and gel as a unit and mark wells on the filter with a pencil (on a clean Whatman). Cut upper left corner of filter, too.
12.) Wash filter 1min. in 2x SSPE. Do not bake filter.
13.) Then prehyb1hour in 37.C: 25 ml H2O
15 ml 20x SSC
2.5 ml 10% Blotto
5 ml 10% SDS
1 ml 0.5 M EDTA
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50 ml
Boil 500 ul denatured salmon sperm DNA (100 ug/ml ?) for 10min, then put on dry ice until frozen. Add just before prehyb starts. Thaw on 68 C. Prehyb in a sealable bag 20 ml per small 12 well gel. Roll off all air bubbles with pipette before sealing bag.
14.) After prehyp open bag and add hot denatured (same as the salmon sperm) probe. (Use elutip purified probe). Add 25ng/20 ml.
15.) Hyb O/N at 37 C (only for oligos / for cDNA it is 68 C).
16.) In am remove filter and wash in 250 ml 2x SSC/0.1% SDS 2x 10min. at RT (oligos)
250 ml 2x SSC/0.5% SDS 2x 15min at RT (cDNA)
250 ml 0.1x SSC/0.1% SDS 2x 30min. at 50 C(oligo)
250 ml 0.3x SSC/0.1% SDS 2x 60min. at 68 C(cDNA
17.) Autoradiograph O/N.
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