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Soriano Lab - Protocols

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southern blot

The following protocol is provided for a 250ml gel, 5-6 mm thick.

  1. 1.Photograph gel, and place in 2 volumes (500 ml) of 0.1N HCl (dilution 1/50). Shake gently for 15 minutes (not more) to depurinate DNA. Pour off HCl.

  2. 2.Transfer gel to 2 volumes of 0.4 N NaOH, shake gently 15 min.

  3. 3.Using gloves, cut two sheets of Whatman 3MM filter paper larger than the gel and two sheets of filter paper and one sheet of HYBOND-XL nylon membrane (Amersham GE Healthcare) to the size of the desired blot.  Do not use Hybond-N+ as new specs make it incompatible with alkaline transfers.

  4. 4.Construct sandwich as follows:

  5. 1.Sponges soaked in 0.4 N NaOH. Change NaOH at periodic intervals.

  6. 2.2 X larger filter paper soaked before hand in 0.4N NaOH. Carefully roll out bubbles with your gloved fingers or a short pipette.

  7. 3.Gel (be sure to remove bubbles from under gel).

  8. 4.Nylon membrane, previously soaked in 0.4N NaOH. Roll out bubbles, this is important as they will interfere with transfer.

  9. 5.2 X filter paper, cut to the same size as the nylon membrane, previously soaked in 0.4 N NaOH. Roll out bubbles.

  10. 6.Use saran wrap to cover all edges of filter paper and sponge, this will prevent wicking.

  11. 7.Stack of paper towels. The saran warp should prevent any contact between the sponges and the paper towels, so that all liquid will go through the gel, ensuring DNA transfer to the nylon membrane.

  12. 8.Place a small weight over paper towels, a small book or catalog should work fine.

  13. 5.Let DNA transfer 3-4 hours. Remove wet paper towels occasionally and add dry towels if necessary.

  14. 6.Neutralize membrane in 2 x SSC, 0.2 M Tris, PH 7.5.

  15. 7.Blot membrane dry and proceed to pre-hybridization (15 minutes to 1 hour) and hybridization. We usually use FBI Buffer [1.5 x SSPE; 10% PEG; 7% SDS; 100ug/ml salmon sperm DNA) at 65oC . Add 1-2 106 cpm/ml denatured probe to a minimal volume of FBI buffer used for the prehybridization. Hybridize for 2 hours to overnight.

  16. 8.Wash filters several times (e.g. 4x) in 1x SSC, 0.1% SDS, or 0.1X SSC, 0.1% SDS according to the stringency desired. Blot filter. While still moist, wrap in saran wrap and proceed to autoradiography.