SOUTHERN BLOTTING OF GENOMIC DNA

1.Digest DNA:

• 1.5 μg DNA in a 25 μI reaction containing BSA

• 3 to 5 units for 5 to 3 hours (aim for 10-fold overdigestion; remember many enzymes will not survive well for more than 3 hr).

• Also prepare molecular weight standard (Lambda plus HindIII); avoid plasmid-derived markers (1 kb ladder,etc.)since it might cross-hybridize with the probe!

2.Run gel:

• Ioad samples onto agarose gel (typically 0.8 to 1.0% agarose in TBE,depending on desired size range).

• run gel at maximum rate of 5 volts per cm.

• run until the bromophenol blue has run ca.9 cm (some DNA will run 1-2 cm below the dye).

• Using gloves,place in staining solution (0.5 to 1 μg/ml ethidium bromide in water,20-40 min.Be careful not to break the gel.

3.Nicking of DNA (to improve transfer of high molecular weight DNA):

• Place the gel (on a plastic plate)in a UV oven.

• Irradiate ca.1200 μJoules.

• Destain 10-30 minutes in water (optional)and photograph.

4.Transfer to nylon membrane:

• WEAR GLOVES when handling blotting membrane,wicks,etc.,since oils on skin will inhibit transfer.WASH OFF powder from gloves before use.

• Soak gel 20-40 minutes in denaturation solution (0.4 M NaOH,0.8 M NaCI)

• Set up capillary blot apparatus (this is one of several transfer methods that can be used for transfer of DNA)as shown on the next page.

• Cut out wicks from Whatman 3 paper using the paper cutter.Setting up the blot will be easiest if one dimension of the wick equals that of the gel.Soak the wick in denaturing solution.Place on plastic sheet suspended on a giass pyrex dish.Use a pipette to rub out any air bubbles.

• CAREFULLY turn over gel and place on top of wick apparatus (transfer is sometimes inhibited by the hardened top surface of gels).Use plastic sheets to help turn over the gel.Use a wet,gloved finger to press out any air bubbies.

• Place a sheet of Hybond N membrane on the gel.Use a piece the same size as the gel.Do not prewet the membrane.Be careful to align the membrane with the gel.Use a pipette to rub out any bubbles.

• Cover with 2 wet sheets of Whatman paper,cut to exactly the same size as the nylon.Cover with 2-4 sheets of dry paper.

• Add "short circuit" prevention device (to make sure that liquid will not bypass the gel and membrane).Use either saran wrap,nescofilm,etc.to cover parts of the wick that might later come into contact with the paper towels.

• Cover with a few inches of blue paper towels,followed by a piastic sheet and weight (1 kg for a 5 x 10 inch gel).

5.Final preparation of membrane (post-blot treatment):

• Let the transfer proceed for 12-18 hr (do not let go longer as the quality of the biot will degrade with time.

• Remove the paper towels and whatman paper from the transfer apparatus,until the membrane is exposed.

• Use a clean blade to notch one side of the membrane to help orient it to the gel (I make a notch on the top of the gel by lane #1).Label the membrane using a pencil.

• Carefully remove the membrane and place,DNA side up,in a dish containing ca.200 ml of 3X SSPE (20X SSPE is 3.6M NaCI,0.2M sodium phosphate,0.02M EDTA pH 7.7).Rock back and forth 10 seconds.Repeat two more times to neutralize the membrane (the alkali transfer covalently bonds the DNA to the membrane; baking or drying is not needed).

• Air dry in a clean and protected piace.Store at room temperature until needed for hybridization.

MAKING PROBES FOR DNA (or RNA)BLOTS

A.Labelling the DNA

The lab currently uses a kit from Amersham.Here is a brief protocol:

1.In screw-cap 1.5 ml tube,place 25 ng DNA in 30 μl water plus 5 μl primer/BSA mix.

2.Boil 5 min; place on ice.

3.Add 10 μl labelling mix.

4.Using a filter-tip pipette tip,add 5 μl (50 μCi)³²P-alpha-dCTP

6.Add 2 uI Klenow.

7.Close the tube.Mix gently by agitating with finger.Spin 10 seconds.

8.Incubate 37℃ 10-30 minutes (for Megaprime kit).

B.Removing unincorporated label

(this helps to eliminates speckle-causing debris,confirms that the probe was OK,and reduces background)

1.Make a minicolumn in a narrow-tipped plastic transfer pipette.

Cut off the bulb (top)

Plug the bottom using a small chunk of sterile,siliconized glass wool.

Support the column in a 2 ml screw-cap tube.

Add gel slurry (Biogel P-60 100-200 mesh in TE [10 mM Tris 8.0,1 mM EDTA] plus 0.2 % SDS).

Add about 2 inch of 66% slurry for a typical column.

Wait for the resin to settle and for the liquid to drain out; discard the liquid.

2.To the DNA labelling reaction (50 μl)add about 20 μl of column loading buffer:

50% glycerol

0.1% bromophenol blue (dark blue)