Southern Blot Analysis(from Baker lab, university of Florida)

DNA Prep

Prepare DNA via your favorite method.You may find a protocol under

Mini Yeast Genomic Prep.

Restriction Digest1.Digest DNA with appropriate restriction enzyme.Digest 10μg of Yeastchromosomal DNA per lane to be run.Digest 0.05 to 0.01μg of plasmid DNA(a massive overkill).2.Allow restriction digest to proceed at 37℃ overnight.

Electrophoresis

1.Seal the gel casting platform at both ends with tape.Prepare and pour a100ml 0.8% agarose in 1X TBE.Insert the comb making sure there are no airbubbles trapped underneath comb and that the all bubbles on the surface ofthe agarose are removed before the gel sets.A16-well comb will allow theloading of approximately 30-35μl of sample.

2.Prepare ³²P labeled 8/HindIII molecular weight standard.Desire 10-20Kcpm to be loaded with 1Fg of cold standard for Ethidium Bromidevisualization.

3.Load gel under fresh 1X TBE and run slowly (100V)to avoid smearing ofsamples until bromophenol blue is near the bottom.It is important to load thegel in an asymmetric pattern,(i.e have the 8/HindIII standard on the rightside only.)this will prevent confusion about loading order on the finalautoradiograph.

4.Stain gel in new ethidium bromide at a maximum concentration of 0.5μg/ml for 15 minutes.

5.Photograph preblot gel for record.

Southern Blot

1.Wash gel in 300ml of 0.25M HCl.Shake gently at room temperature on platform shaker for 30 minutes.Depurinates DNA to facilitate transfer of large DNA.

2.Wash gel in 500ml of 0.5M NaOH,1.0 M NaCl.Shake gently at room temperature on platform shaker for 20 minutes.Denatures DNA for probe access.

3.Prepare 2 liters of 0.025M NaPO₄ pH6.5 transfer buffer (25ml 0.5M Na₂HPO₄,75ml 0.5M NaH₂ PO₄ / 2 liter).

4.Cut Hybond N to the size of the gel and wet with transfer buffer (Gloves a must).

5.Cut 4 pieces of Whatman 3MM paper to the same size as the gel and one twice as long to be used as a wick.

6.Set up capillary blot as per Maniatis.Transfer for at least 6 hours,normally we allow the transfer to proceed overnight.

7.Place gel support in Pyrex dish,add transfer buffer.Place long Whatman 3MM (wick)over support so that the edges are submerged in the transfer buffer.Roll out any air bubbles by gently rolling a glass tube over the surface.

8.Place 2 pieces of Whatman 3MM,same size of gel,wet with transfer buffer.Roll out any bubbles.

9.Place gel gently on top of Whatman 3MM.Very gently with wet gloves roll out bubbles.

10.Cut four strips of saran wrap and place over the edges of the gel.This is to prevent the buffer from "short -circuiting".So that the buffer flows through rather than around the gel.

11.Place Hybond N membrane exactly over the gel.Very gently squeeze air bubbles by gently rolling a glass tube over the surface.

12.Place 2 pieces of Whatman 3MM,same size of gel,wet with transfer buffer over the Hybond N .Try to avoid getting air bubbles under the membrane;remove any by carefully rolling a glass tube over the surface.

13.Cut paper towels to the same size as the membrane and stack these on top of Whatman 3MM papers to a height of about 10cm.

14.Lay a glass plate on top of the structure and place a weight on top to hold everything in place.

Disassemble the transfer pyramid

1.Once the blot is complete,disassemble the transfer pyramid and recover your membrane.Remove left bottom corner of blot,to mark DNA side.

2.Cross-link membrane by placing blot on plastic wrap,DNA-side down,on transilluminator and illuminating at full power for 5 minutes.

3.Stain transferred gel in EtBr and photograph to ensure complete blotting has been done.(Labeled Lambda should now be on filter and you may test it with the Geiger counter.)

4.Wash cross linked filter in 250ml of 0.1X SSC,0.5% SDS at 65℃ for 45 minutes to remove residual agarose.This step is critical,do not omit or the background will be very high after hibridizing.

Hybridization

(Church and Gilbert,1984)

(1).Prehybridization

①.Once membrane has been cross linked and washed,roll and place into glass cylinder so that it sticks completely to the walls.

②.Add 5ml of Hybridization buffer to small cylinder (8ml for large cylinder)

③.Prehybridize at 65℃ for at least 15 minutes and up to 2 hrs.

④.Write in your notes the temperature and actual time of prehybridization.

(2).Hybridization

①.Decant prehybridizing buffer and pour probe into cylinder prepared as indicated under probe.

②.Hybridize at 65℃ overnight.

③.Make a note of temperature and total time of hybridization.

(3).Washing

Washing is done at 65℃ with prewarmed washing solution.